大肠杆菌K12 GlcNAc-1-P尿苷转移酶的研究
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摘要
以寡糖和糖复合物(糖脂、糖蛋白等)形式存在的糖类化合物是发现于所有生命体的重要生物聚合物,它们在众多复杂生物过程中发挥着不可替代的重要作用。糖链结构形式的特定改变与特定的病理状态(如癌症和炎症)机密相关,这显示了糖链在临床诊断中的应用潜力,以及作为药物开发靶点的可能性。
自然界中糖链的生物合成是由糖基转移酶催化进行的,它们将相应的糖核苷酸上特定的单糖转移到一个糖基受体的特定羟基基团上,形成糖苷键的共价连接。由于其在高效合成高度空间特异性和立体化学特异性的糖苷键方面展现出的优势,利用糖基转移酶催化糖链合成已经成为利用有机化学手段合成糖链的有效替代途径。有机合成手段可以为天然化合物提供多样性的衍生物,为药物开发研究提供更多选择性靶点。将生物酶法和化学法结合的化学-酶法合成,是近年来广泛应用于糖生物学研究领域的一个重要手段,有机合成和生物酶法优势互补,成为目前糖生物学和糖化学研究领域的一个充满生机活力的研究方法和手段。
糖核苷酸(nucleotide sugars)亦称为活性糖(active sugars),在化学结构上是单糖的还原端和核苷一磷酸或二磷酸的末端磷酸基团结合形成的化合物。糖核苷酸的生理意义主要包括:1.通过糖核苷酸之间的相互转化,产生一系列糖基转移酶催化反应所必须的活性糖;2.在糖苷和多糖的生物合成过程中,作为糖的供体,是糖单元合成的前体。
UDP-GlcNAc是细胞内的一种重要的氨基糖供体,是细胞内多种细胞分子合成的前体物质。这些细胞内分子主要包括细胞壁肽聚糖、脂多糖、肠杆菌科细菌表面共同抗原、几丁寡糖、GPI锚、糖胺聚糖和糖蛋白等。
生物体内UDP-GlcNAc的合成,都以己糖代谢途径的中间产物果糖-6-磷酸(Fructose-6-P)为起始底物,在多种酶的协同催化作用下,最终合成UDP-GlcNAc。根据合成过程中催化反应的顺序及合成途径所涉及酶的来源不同,分为真核UDP-GlcNAc合成途径和原核UDP-GlcNAc合成途径。两种合成途径的主要区别在于氨基葡萄糖-6-磷酸(GlcN-6-P)的乙酰化和异构化反应的先后顺序不同。真核合成途径中,GlcN-6-P先在乙酰转移酶的作用下,生成乙酰氨基葡萄糖-6-磷酸(GlcNAc-6-P),然后再由异构酶作用,生成乙酰氨基葡萄糖-1-磷酸(GlcNAc-1-P);而原核合成途径中,GlcN-6-P先异构化为氨基葡萄糖-1-磷酸(GlcN-1-P),然后再在乙酰转移酶催化下生成GlcNAc-1-P。
本论文以大肠杆菌K12来源的GlmU为研究对象,对GlmU和GlmU的N-端结构域GlmU-Tr229的底物广泛性进行了系统研究。除了以揭示它们的底物适应性、酶的动力学性质和催化机理为目的的生化研究,本论文还通过体外小量合成反应验证了GlmU在氨基糖核苷酸合成中的潜在应用价值;本论文还就GlmU突变体进行了初步研究,取得了一定的成果。
论文第二章我们克隆了来自Escherichia coli K12的GlcNAc-1-P尿苷转移酶(GlmU),IPTG诱导GlmU蛋白在E coli BL21(DE3)中表达,带有N-端His标签的GlmU蛋白经过Ni-NTA纯化,SDS-PAGE电泳结果显示,GlmU纯度达到90%以上,GlmU单体分子的表观分子量约为50kDa,与理论推导值基本一致。
论文研究了底物GlcNAc-1-P的C2位化学修饰对GlmU催化反应的影响。分别使用了C2位是羟基(Glc),C2位为乙酰氨基(GlcNAc)和C2位为有叠氮基修饰(GlcNAcZ)的三种不同糖-1-磷酸衍生物。实验结果发现,GlmU对三种糖-1-磷酸有不同的催化活性,以UTP为核苷三磷酸供体的反应,使用GlcNAcZ-1-P和Glc-1-P的反应得率分别是以GlcNAc-1-P为底物时反应得率的86%和30%。以前的相关研究表明,GlcNAc-1-P的乙酰氨基与酶分子Thr82和Glu154残基以氢键相互作用。Glc-1-P反应转化率的降低进一步证明了Thr82和Glu154残基在糖-1-磷酸识别中的重要作用。相反,GlcNAcZ-1-P反应转化率没有显著变化的结果表明GlmU能够耐受乙酰氨基上的修饰。
第三章结合E.coli来源的GlmU已有晶体结构,克隆并构建了来自E.coli K12的GlmU的N-端结构域(GlcNAc-1-P尿苷转移酶结构域,GlmU-Tr229),蛋白表达水平为55mg/L。为系统研究GlmU-Tr229对不同核苷三磷酸的底物耐受性,研究中使用了GlcNAc-1-P做催化反应底物,使用了12种不同的核苷三磷酸(NTP),利用毛细管电泳检测NDP-GlcNAc的合成情况。NDP-GlcNAc产率结果显示,GlmU-Tr229对不同核苷三磷酸具有一个底物耐受顺序:UTP＞dUTP＞dTTP＞＞CTP＞dATP/dm~6ATP,这结果表明GlmU对嘧啶核苷酸的利用程度要优于对嘌呤核苷酸的利用。
GlmU蛋白晶体研究结果已经阐明GlmU N-端催化结构域被两个突出结构包围:第一个突出结构(Asn3-Val111及His216-227),主要参与识别和结合UDP-GlcNAc的核苷部分;第二个突出结构(Glu112-Val215)则主要是与糖核苷酸中的糖结构相互作用有关。尿嘧啶通过尿嘧啶环N3与Gln76之间及4位羰基氧与Gln76、Gly81之间形成的氢键被识别和结合。核苷中的核糖结构,主要是通过核糖2位的OH基团与Gly14之间的氢键作用。我们对GlmU-Tr229的研究结果表明,GlmU N-端催化结构域有非常显著的底物耐受性,对核糖2位的修饰(dUTP)或者是对尿嘧啶环C5修饰(dTTP)都不会显著影响N-端催化结构域的活性。这些结果表明,核糖2位OH与Gly14之间的氢键在底物识别过程中不是必需的。
为验证利用重组GlmU-Tr229合成UDP-GlcNAc衍生物的应用前景,我们在毫克水平上合成了UDP-GlcNAc的两种衍生物:dUDP-GlcNAc和UDP-GlcNAcZ。并使用mono Q离子交换层析和P2分子筛凝胶对糖核苷酸产物进行分离纯化,最终使用ESI-MS和NMR对得到的dUDP-GlcNAc(5.1mg,57.6%)和UDP-GlcNAcZ(4.3mg,44.4%)进行了定性分析。
本论文对GlmU-Tr229生化性质进行了细致的研究,阐述了GlmU-Tr229催化反应需要依赖金属离子做辅助因子,GlmU-Tr229对金属离子的依赖性为:CO~(2+)＞Mn~(2+)＞Mg~(2+)＞＞Zn~(2+)/Cu~(2+)/Ni~(2+)＞EDTA;在以Mg~(2+)为辅助因子的催化反应体系中,5 mM Mg~(2+)是最适的金属离子浓度,研究了pH对GlmU-Tr229催化活性的影响,GlmU-Tr229最适pH是7.5,pH6.5-8.5范围内都能够催化反应的进行。我们还对催化反应中副产物焦磷酸的反馈抑制作用进行了研究,通过在反应体系中加入焦磷酸水解酶,将反应体系中累积的焦磷酸水解为无机磷酸根,以UTP和GlcNAc-1-P为底物的催化反应,转化率由原来的65%提高到95%。
第四章中,我们利用一种六碳糖激酶(NahK),以ATP和GlcNAc为底物,体外酶促反应合成并分离纯化获得GlcNAc-1-P。探索了一条体外利用NahK合成GlmU反应前体物质GlcNAc-1-P的新途径,解决了GlmU酶学研究底物供给不足的瓶颈。同时,为深入研究GlmU-Tr229底物特异性,我们对GlmU-Tr229的Gln76和Gly81氨基酸位点进行了定点突变研究,并对其中一个突变体Q76E进行了酶学性质的研究。通过毛细管电泳检测发现Q76E的突变引起了GlmU-Tr229对NTP底物利用情况的改变,天然底物UTP利用率大幅降低,而非天然底物CTP的反应转化率达50%以上,这部分工作正在进行中。
总之,本工作提供了一条切实可行的合成UDP-GlcNAc类似物的途径,可以用于乙酰氨基葡萄糖转移酶和细胞内多种细胞组分的合成途径研究。对大肠杆菌K12来源的GlmU和GlmU-Tr229底物广泛性的系统研究有助于我们提高对氨基糖核苷酸合成基础知识的认识。
Glycans in the form of oligosaccharides and glycoconjugates(glycolipids,glycoproteins,etc ) are vital biopolymers found in organisms across all domains of life.They play critical roles in mediation of numerous complex biological processes.Specific changes in glycan profiles have been associated with certain disease states such as cancer and inflammation,illustrating the potential of using glycans in clinical diagnosis and perhaps as targets to develop therapeutics.
The biosynthesis of saccharides in nature uses glycosyltransferase.They transfer a given monosaccharide from the corresponding sugar nucleotide(sugar donor) to a specific hydroxyl group of a sugar acceptor.With obvious advantages of achieving high regio- and stereochemistry of glycosidic bonds,often in a high efficient manner,glycosyltransferase-catalyzed enzymatic synthesis of saccharides becomes an attractive and powerful alternative to the chemical synthesis. Oranic synthesis could give targets in therapeutics development by synthesizing complex analogs using nature compounds.Chem-enzyme synthesis becomes an important research way and mean in the research field of Glycobiology and Glcochemistry by combining bio-enzyme and organic methods exploiting their own parricular advantages for mutual benefit.
Nucleotide sugars,also call active sugars,are compounds which are synthesized using reducing end of monosaccharides and the ending phosphate group of nucleotide monophosphate or bisphosphate.The biological importance for those compounds:1 to product necessary active saccharide for glycosyltransferase reaction by transformation among nucleotide sugars;2 to act as glycosyl donors in glycan and polysaccharides biosynthesis pathway.
Uridine 5'-diphosphate N-acetylglucosamine(UDP-GlcNAc),the key cytoplasmic amino nucleotide sugar,is an essential precursor of the biosynthesis of various cell components including cell wall peptidoglycan,lipopolysaccharides,enterobacterial common antigens,chitin, glycosylphophatidylinositol anchors,glycosaminoglycans and glycoproteins.
UDP-GlcNAc was synthesized by a multi-enzymatic reaciton using fructose-6-P which produced during glycolysis acts as starting substrates in in vivo UDP-GlcNAc synthesis pathway. Two variations of this pathway exist,one in eukaryotes and one in prokaryotes.The main difference between two pathways is the acetylation and isomerization order for glucosamine-6-P. In the eukaryotic pathway,fructose-6-phosphate produced during glycolysis is transaminated and isomerized to glucosamine-6-phosphate,followed by acetylation to GlcNAc-6-phosphate, isomerization to GlcNAc-1-phosphate.In prokaryotes,glucosamine-6-phosphate is isomerized to glucosamine-1-phosphate,followed by its N-acetylation to N-acetyl-D-glucosamine-1-phosphate.
The aim of this thesis is exploring the bifunctional GlcNAc-1-P uridyltransferase (GlcNAc-1-P pyrophosphorylase,GlmU) and its uridyltransferase domain(GlmU-Tr229) from Escherichi coli K12 for systematic study on broad substrate specificity.In addition to the biochemical study towards the illumination of their substrate adaptability,enzymatic dynamics and catalytic mechanism,the synthetic application of this enzyme was also elucidated by the synthesis of two amino nucleotide sugars in small scale.Pilot study on the GlmU site-directed muation were also carried out and got some progress.
In chapter 2,GlmU was cloned from the genome of E.coli K12 and was over-expressed in E. coli BL21(DE3).The recombinant His-tagged GlmU protein was purified by Ni-NTA column. SDS-PAGE showed that the purity of GlmU protein was more than 90%,the apparent molecular weight of the GlmU protein was about 50 kDa,consistent with the calculated mass.
The effect of N-acetyl group GlcNAc on GlmU enzymatic reaction was well investigated using Glc-1-P(C2 group OH),GlcNAc-1-P(C2 group N-acetyl) and GlcNAcZ-1-P(C2 group N-adize-acetyl group).GlmU exhibited activity for all three sugar-1-Ps with diverse yields.The yields for GlcNAcZ-1-P decreased to 86%of that for GlcNAc-1-P with UTP as pyrophosphate donor.While the conversion efficiencies for Glc-1-P which lacks the C2 N-acetyl group dropped to around 30%of this for GlcNAc-1-P.Previous study suggested that GlcNAc-1-P interacted with the enzyme by hydrogen bonds between the N-acetyl arm and Thr82 and Glu154.The significant decline of GlmU activity for Glc-1-P compared with that for GlcNAc-1-P was consistent with the structure study,which demonstrated the important role of Thr82 and Glu154 residues in sugar-1-P recognition.In contrast,GlmU-Tr229 had better tolerance for the modifications on the N-acyl group,as indicated by the result for GlcNAcZ-1-P.
In chapter 3,bifunctional GlmU N-terminal uridyltransferase domain(GlmU-Tr229) was cloned and expressed in E.coli BL21(DE3) according to the crystal structure of GlmU with a 55 mg per Liter souble protein expression level.To investigate the substrate specificity towards NTP, GlcNAc-1-P was used as the sugar-1-P substrate.Twelve NTPs were included into the reaction system,respectively.Capillary electrophoresis(CE) was used to detect the formation of NDP-GlcNAcs.The yields of NDP-GlcNAcs indicated a substrate tolerance of GlmU-Tr229 for NTPs in the following order:UTP＞dUTP＞dTTP＞＞CTP＞dATP/dm~6ATP,suggesting that GlmU-Tr229 prefers pyrimidine nucleotide than purine nucleotide.
The crystal structure of GlmU showed that the active site pocket of the pyrophosphorylase domain was bound by two lobes.The first lobe(residues Asn3-Val111 and His216-227) included strandsβ1-β4,which interacted primarily with the nucleotide moiety of UDP-GlcNAc.The second lobe(residues Glu112-Val215) included strandsβ5-β7,which interacted primarily with GlcNAc portion of the sugar nucleotide.Uracil base was recognized by a hydrogen bond between its ring N3 and Gln76 residue,and by two hydrogen bonds between its exocyclic oxygen O4 and Gly81 and Gln76,respectively.The ribose group was recognized by a hydrogen bond between its 2'-hydroxyl group and Gly14.Our results indicated that pyrophosphorylase domain of GlmU had a notable substrate tolerance,small changes on ribose 2'-hydroxyl group(dUTP) or uracil base C5 (dTTP) do not affect the pyrophosphorylase activity.The hydrogen bond between ribose group 2'-hydroxyl group and Gly14 was not critical for substrate recognition.The first lobe which contained the pyrophosphorylase active site was suitable to bear the existence of a methyl group in uracil base C5.
To demonstrate the application of the recombinant GlmU-Tr229 for the synthesis of UDP-GlcNAc analogs,we performed a synthetic reaction in multiple mg scale for dUDP-GlcNAc and UDP-GlcNAcZ.Mono Q ion-exchange column(GE Healthcare) was used to isolate the products from the reaction mixture.The products were further desalted by P2 gel filtration column (Bio-rad).The isolated dUDP-GlcNAc(5.1 mg,57.6%) and UDP-GlcNAcZ(4.3 mg,44.4%) were identified by ESI-MS and NMR spectroscopy.
We also investigated biochemical characters of GlmU-Tr229.Our results demonstrated that divalent metal was necessary for GlcNAc-1-P uridyltransfer reaction,which could be used as GlmU cofactor.The dependent relationship of divalent metal ranked:Co~(2+)＞Mn~(2+)＞Mg~(2+)＞＞Zn~(2+) /Cu~(2+)/Ni~(2+)＞EDTA.The optimal divalent metal cocentration for the reaction was 5 mM when Mg~(2+) was used as cofactor.GlmU-Tr229 catalyzed GlcNAc-1-P uridyltransfer reaction under pH6.5-8.5 with an optimal pH 7.5.In addition,the product inhibition effect of the reaction byproduct inorganic pyrophosphate(IPP) was studied.Pyrophosphatase which catalyzes the hydrolysis of IPP to two orthophosphates was omitted to the reaction mixture.UDP-GlcNAc conversion ratio decreased to 65%in contrast with 95%at maximum when pyrophosphatase existed in the reaction.The result showed inhibition of IPP to the reaction and hydrolysis of IPP by pyrophosphatase could facilitate the uridyl transfer reaction.
In chaper 4,GlcNAc-1-P was synthesized by a N-acetylhexosamine 1-kinse from Bifidobacterium longum using ATP and GlcNAc as substrates in in vitro reaction.The product GlcNAc-1-P was then separated by organic method using a sialic gel column.Lack of GlcNAc-1-P could be settled using this synthsis pathway in GlmU enzymatic study.
To further study on nucleotide triphosphate substrate specificity of the E.coli K12 GlmU-Tr229,site-directed mutaions were done in residues Gln76 and Gly81 of GlmU-Tr229. Biochemical characters of one of the mutations GlmU-Tr229 Q76E was well studied using UTP and CTP as nucleotide triphosphate,respectively.Capillary electrophoresis profiles showed that GlmU-Tr229 Q76E had a notable change in nucleotide triphosphate substrate specificity.CTP could be recognized and used in the formation of CDP-GlcNAc with a yield above 50%.However, the conversion ratio for UTP reactions was decreased to 50%in contrast with 95%at mixmum.in GlmU reaction.This work is still ongoing.
In sumary,this work provided a feasible approach for the synthesis of UDP-GlcNAc analogs which can be used to study GlcNAc-transferase reaction and various biosynthesis pathways for cell components.The systematic study on the broad substrate specificity of GlmU and GlmU-Tr229 from E.coli K12 could help us to enhance the basic knowledge of enzyme realted to amio-sugar nulceotide synthesis.
自然界中糖链的生物合成是由糖基转移酶催化进行的,它们将相应的糖核苷酸上特定的单糖转移到一个糖基受体的特定羟基基团上,形成糖苷键的共价连接。由于其在高效合成高度空间特异性和立体化学特异性的糖苷键方面展现出的优势,利用糖基转移酶催化糖链合成已经成为利用有机化学手段合成糖链的有效替代途径。有机合成手段可以为天然化合物提供多样性的衍生物,为药物开发研究提供更多选择性靶点。将生物酶法和化学法结合的化学-酶法合成,是近年来广泛应用于糖生物学研究领域的一个重要手段,有机合成和生物酶法优势互补,成为目前糖生物学和糖化学研究领域的一个充满生机活力的研究方法和手段。
糖核苷酸(nucleotide sugars)亦称为活性糖(active sugars),在化学结构上是单糖的还原端和核苷一磷酸或二磷酸的末端磷酸基团结合形成的化合物。糖核苷酸的生理意义主要包括:1.通过糖核苷酸之间的相互转化,产生一系列糖基转移酶催化反应所必须的活性糖;2.在糖苷和多糖的生物合成过程中,作为糖的供体,是糖单元合成的前体。
UDP-GlcNAc是细胞内的一种重要的氨基糖供体,是细胞内多种细胞分子合成的前体物质。这些细胞内分子主要包括细胞壁肽聚糖、脂多糖、肠杆菌科细菌表面共同抗原、几丁寡糖、GPI锚、糖胺聚糖和糖蛋白等。
生物体内UDP-GlcNAc的合成,都以己糖代谢途径的中间产物果糖-6-磷酸(Fructose-6-P)为起始底物,在多种酶的协同催化作用下,最终合成UDP-GlcNAc。根据合成过程中催化反应的顺序及合成途径所涉及酶的来源不同,分为真核UDP-GlcNAc合成途径和原核UDP-GlcNAc合成途径。两种合成途径的主要区别在于氨基葡萄糖-6-磷酸(GlcN-6-P)的乙酰化和异构化反应的先后顺序不同。真核合成途径中,GlcN-6-P先在乙酰转移酶的作用下,生成乙酰氨基葡萄糖-6-磷酸(GlcNAc-6-P),然后再由异构酶作用,生成乙酰氨基葡萄糖-1-磷酸(GlcNAc-1-P);而原核合成途径中,GlcN-6-P先异构化为氨基葡萄糖-1-磷酸(GlcN-1-P),然后再在乙酰转移酶催化下生成GlcNAc-1-P。
本论文以大肠杆菌K12来源的GlmU为研究对象,对GlmU和GlmU的N-端结构域GlmU-Tr229的底物广泛性进行了系统研究。除了以揭示它们的底物适应性、酶的动力学性质和催化机理为目的的生化研究,本论文还通过体外小量合成反应验证了GlmU在氨基糖核苷酸合成中的潜在应用价值;本论文还就GlmU突变体进行了初步研究,取得了一定的成果。
论文第二章我们克隆了来自Escherichia coli K12的GlcNAc-1-P尿苷转移酶(GlmU),IPTG诱导GlmU蛋白在E coli BL21(DE3)中表达,带有N-端His标签的GlmU蛋白经过Ni-NTA纯化,SDS-PAGE电泳结果显示,GlmU纯度达到90%以上,GlmU单体分子的表观分子量约为50kDa,与理论推导值基本一致。
论文研究了底物GlcNAc-1-P的C2位化学修饰对GlmU催化反应的影响。分别使用了C2位是羟基(Glc),C2位为乙酰氨基(GlcNAc)和C2位为有叠氮基修饰(GlcNAcZ)的三种不同糖-1-磷酸衍生物。实验结果发现,GlmU对三种糖-1-磷酸有不同的催化活性,以UTP为核苷三磷酸供体的反应,使用GlcNAcZ-1-P和Glc-1-P的反应得率分别是以GlcNAc-1-P为底物时反应得率的86%和30%。以前的相关研究表明,GlcNAc-1-P的乙酰氨基与酶分子Thr82和Glu154残基以氢键相互作用。Glc-1-P反应转化率的降低进一步证明了Thr82和Glu154残基在糖-1-磷酸识别中的重要作用。相反,GlcNAcZ-1-P反应转化率没有显著变化的结果表明GlmU能够耐受乙酰氨基上的修饰。
第三章结合E.coli来源的GlmU已有晶体结构,克隆并构建了来自E.coli K12的GlmU的N-端结构域(GlcNAc-1-P尿苷转移酶结构域,GlmU-Tr229),蛋白表达水平为55mg/L。为系统研究GlmU-Tr229对不同核苷三磷酸的底物耐受性,研究中使用了GlcNAc-1-P做催化反应底物,使用了12种不同的核苷三磷酸(NTP),利用毛细管电泳检测NDP-GlcNAc的合成情况。NDP-GlcNAc产率结果显示,GlmU-Tr229对不同核苷三磷酸具有一个底物耐受顺序:UTP＞dUTP＞dTTP＞＞CTP＞dATP/dm~6ATP,这结果表明GlmU对嘧啶核苷酸的利用程度要优于对嘌呤核苷酸的利用。
GlmU蛋白晶体研究结果已经阐明GlmU N-端催化结构域被两个突出结构包围:第一个突出结构(Asn3-Val111及His216-227),主要参与识别和结合UDP-GlcNAc的核苷部分;第二个突出结构(Glu112-Val215)则主要是与糖核苷酸中的糖结构相互作用有关。尿嘧啶通过尿嘧啶环N3与Gln76之间及4位羰基氧与Gln76、Gly81之间形成的氢键被识别和结合。核苷中的核糖结构,主要是通过核糖2位的OH基团与Gly14之间的氢键作用。我们对GlmU-Tr229的研究结果表明,GlmU N-端催化结构域有非常显著的底物耐受性,对核糖2位的修饰(dUTP)或者是对尿嘧啶环C5修饰(dTTP)都不会显著影响N-端催化结构域的活性。这些结果表明,核糖2位OH与Gly14之间的氢键在底物识别过程中不是必需的。
为验证利用重组GlmU-Tr229合成UDP-GlcNAc衍生物的应用前景,我们在毫克水平上合成了UDP-GlcNAc的两种衍生物:dUDP-GlcNAc和UDP-GlcNAcZ。并使用mono Q离子交换层析和P2分子筛凝胶对糖核苷酸产物进行分离纯化,最终使用ESI-MS和NMR对得到的dUDP-GlcNAc(5.1mg,57.6%)和UDP-GlcNAcZ(4.3mg,44.4%)进行了定性分析。
本论文对GlmU-Tr229生化性质进行了细致的研究,阐述了GlmU-Tr229催化反应需要依赖金属离子做辅助因子,GlmU-Tr229对金属离子的依赖性为:CO~(2+)＞Mn~(2+)＞Mg~(2+)＞＞Zn~(2+)/Cu~(2+)/Ni~(2+)＞EDTA;在以Mg~(2+)为辅助因子的催化反应体系中,5 mM Mg~(2+)是最适的金属离子浓度,研究了pH对GlmU-Tr229催化活性的影响,GlmU-Tr229最适pH是7.5,pH6.5-8.5范围内都能够催化反应的进行。我们还对催化反应中副产物焦磷酸的反馈抑制作用进行了研究,通过在反应体系中加入焦磷酸水解酶,将反应体系中累积的焦磷酸水解为无机磷酸根,以UTP和GlcNAc-1-P为底物的催化反应,转化率由原来的65%提高到95%。
第四章中,我们利用一种六碳糖激酶(NahK),以ATP和GlcNAc为底物,体外酶促反应合成并分离纯化获得GlcNAc-1-P。探索了一条体外利用NahK合成GlmU反应前体物质GlcNAc-1-P的新途径,解决了GlmU酶学研究底物供给不足的瓶颈。同时,为深入研究GlmU-Tr229底物特异性,我们对GlmU-Tr229的Gln76和Gly81氨基酸位点进行了定点突变研究,并对其中一个突变体Q76E进行了酶学性质的研究。通过毛细管电泳检测发现Q76E的突变引起了GlmU-Tr229对NTP底物利用情况的改变,天然底物UTP利用率大幅降低,而非天然底物CTP的反应转化率达50%以上,这部分工作正在进行中。
总之,本工作提供了一条切实可行的合成UDP-GlcNAc类似物的途径,可以用于乙酰氨基葡萄糖转移酶和细胞内多种细胞组分的合成途径研究。对大肠杆菌K12来源的GlmU和GlmU-Tr229底物广泛性的系统研究有助于我们提高对氨基糖核苷酸合成基础知识的认识。
Glycans in the form of oligosaccharides and glycoconjugates(glycolipids,glycoproteins,etc ) are vital biopolymers found in organisms across all domains of life.They play critical roles in mediation of numerous complex biological processes.Specific changes in glycan profiles have been associated with certain disease states such as cancer and inflammation,illustrating the potential of using glycans in clinical diagnosis and perhaps as targets to develop therapeutics.
The biosynthesis of saccharides in nature uses glycosyltransferase.They transfer a given monosaccharide from the corresponding sugar nucleotide(sugar donor) to a specific hydroxyl group of a sugar acceptor.With obvious advantages of achieving high regio- and stereochemistry of glycosidic bonds,often in a high efficient manner,glycosyltransferase-catalyzed enzymatic synthesis of saccharides becomes an attractive and powerful alternative to the chemical synthesis. Oranic synthesis could give targets in therapeutics development by synthesizing complex analogs using nature compounds.Chem-enzyme synthesis becomes an important research way and mean in the research field of Glycobiology and Glcochemistry by combining bio-enzyme and organic methods exploiting their own parricular advantages for mutual benefit.
Nucleotide sugars,also call active sugars,are compounds which are synthesized using reducing end of monosaccharides and the ending phosphate group of nucleotide monophosphate or bisphosphate.The biological importance for those compounds:1 to product necessary active saccharide for glycosyltransferase reaction by transformation among nucleotide sugars;2 to act as glycosyl donors in glycan and polysaccharides biosynthesis pathway.
Uridine 5'-diphosphate N-acetylglucosamine(UDP-GlcNAc),the key cytoplasmic amino nucleotide sugar,is an essential precursor of the biosynthesis of various cell components including cell wall peptidoglycan,lipopolysaccharides,enterobacterial common antigens,chitin, glycosylphophatidylinositol anchors,glycosaminoglycans and glycoproteins.
UDP-GlcNAc was synthesized by a multi-enzymatic reaciton using fructose-6-P which produced during glycolysis acts as starting substrates in in vivo UDP-GlcNAc synthesis pathway. Two variations of this pathway exist,one in eukaryotes and one in prokaryotes.The main difference between two pathways is the acetylation and isomerization order for glucosamine-6-P. In the eukaryotic pathway,fructose-6-phosphate produced during glycolysis is transaminated and isomerized to glucosamine-6-phosphate,followed by acetylation to GlcNAc-6-phosphate, isomerization to GlcNAc-1-phosphate.In prokaryotes,glucosamine-6-phosphate is isomerized to glucosamine-1-phosphate,followed by its N-acetylation to N-acetyl-D-glucosamine-1-phosphate.
The aim of this thesis is exploring the bifunctional GlcNAc-1-P uridyltransferase (GlcNAc-1-P pyrophosphorylase,GlmU) and its uridyltransferase domain(GlmU-Tr229) from Escherichi coli K12 for systematic study on broad substrate specificity.In addition to the biochemical study towards the illumination of their substrate adaptability,enzymatic dynamics and catalytic mechanism,the synthetic application of this enzyme was also elucidated by the synthesis of two amino nucleotide sugars in small scale.Pilot study on the GlmU site-directed muation were also carried out and got some progress.
In chapter 2,GlmU was cloned from the genome of E.coli K12 and was over-expressed in E. coli BL21(DE3).The recombinant His-tagged GlmU protein was purified by Ni-NTA column. SDS-PAGE showed that the purity of GlmU protein was more than 90%,the apparent molecular weight of the GlmU protein was about 50 kDa,consistent with the calculated mass.
The effect of N-acetyl group GlcNAc on GlmU enzymatic reaction was well investigated using Glc-1-P(C2 group OH),GlcNAc-1-P(C2 group N-acetyl) and GlcNAcZ-1-P(C2 group N-adize-acetyl group).GlmU exhibited activity for all three sugar-1-Ps with diverse yields.The yields for GlcNAcZ-1-P decreased to 86%of that for GlcNAc-1-P with UTP as pyrophosphate donor.While the conversion efficiencies for Glc-1-P which lacks the C2 N-acetyl group dropped to around 30%of this for GlcNAc-1-P.Previous study suggested that GlcNAc-1-P interacted with the enzyme by hydrogen bonds between the N-acetyl arm and Thr82 and Glu154.The significant decline of GlmU activity for Glc-1-P compared with that for GlcNAc-1-P was consistent with the structure study,which demonstrated the important role of Thr82 and Glu154 residues in sugar-1-P recognition.In contrast,GlmU-Tr229 had better tolerance for the modifications on the N-acyl group,as indicated by the result for GlcNAcZ-1-P.
In chapter 3,bifunctional GlmU N-terminal uridyltransferase domain(GlmU-Tr229) was cloned and expressed in E.coli BL21(DE3) according to the crystal structure of GlmU with a 55 mg per Liter souble protein expression level.To investigate the substrate specificity towards NTP, GlcNAc-1-P was used as the sugar-1-P substrate.Twelve NTPs were included into the reaction system,respectively.Capillary electrophoresis(CE) was used to detect the formation of NDP-GlcNAcs.The yields of NDP-GlcNAcs indicated a substrate tolerance of GlmU-Tr229 for NTPs in the following order:UTP＞dUTP＞dTTP＞＞CTP＞dATP/dm~6ATP,suggesting that GlmU-Tr229 prefers pyrimidine nucleotide than purine nucleotide.
The crystal structure of GlmU showed that the active site pocket of the pyrophosphorylase domain was bound by two lobes.The first lobe(residues Asn3-Val111 and His216-227) included strandsβ1-β4,which interacted primarily with the nucleotide moiety of UDP-GlcNAc.The second lobe(residues Glu112-Val215) included strandsβ5-β7,which interacted primarily with GlcNAc portion of the sugar nucleotide.Uracil base was recognized by a hydrogen bond between its ring N3 and Gln76 residue,and by two hydrogen bonds between its exocyclic oxygen O4 and Gly81 and Gln76,respectively.The ribose group was recognized by a hydrogen bond between its 2'-hydroxyl group and Gly14.Our results indicated that pyrophosphorylase domain of GlmU had a notable substrate tolerance,small changes on ribose 2'-hydroxyl group(dUTP) or uracil base C5 (dTTP) do not affect the pyrophosphorylase activity.The hydrogen bond between ribose group 2'-hydroxyl group and Gly14 was not critical for substrate recognition.The first lobe which contained the pyrophosphorylase active site was suitable to bear the existence of a methyl group in uracil base C5.
To demonstrate the application of the recombinant GlmU-Tr229 for the synthesis of UDP-GlcNAc analogs,we performed a synthetic reaction in multiple mg scale for dUDP-GlcNAc and UDP-GlcNAcZ.Mono Q ion-exchange column(GE Healthcare) was used to isolate the products from the reaction mixture.The products were further desalted by P2 gel filtration column (Bio-rad).The isolated dUDP-GlcNAc(5.1 mg,57.6%) and UDP-GlcNAcZ(4.3 mg,44.4%) were identified by ESI-MS and NMR spectroscopy.
We also investigated biochemical characters of GlmU-Tr229.Our results demonstrated that divalent metal was necessary for GlcNAc-1-P uridyltransfer reaction,which could be used as GlmU cofactor.The dependent relationship of divalent metal ranked:Co~(2+)＞Mn~(2+)＞Mg~(2+)＞＞Zn~(2+) /Cu~(2+)/Ni~(2+)＞EDTA.The optimal divalent metal cocentration for the reaction was 5 mM when Mg~(2+) was used as cofactor.GlmU-Tr229 catalyzed GlcNAc-1-P uridyltransfer reaction under pH6.5-8.5 with an optimal pH 7.5.In addition,the product inhibition effect of the reaction byproduct inorganic pyrophosphate(IPP) was studied.Pyrophosphatase which catalyzes the hydrolysis of IPP to two orthophosphates was omitted to the reaction mixture.UDP-GlcNAc conversion ratio decreased to 65%in contrast with 95%at maximum when pyrophosphatase existed in the reaction.The result showed inhibition of IPP to the reaction and hydrolysis of IPP by pyrophosphatase could facilitate the uridyl transfer reaction.
In chaper 4,GlcNAc-1-P was synthesized by a N-acetylhexosamine 1-kinse from Bifidobacterium longum using ATP and GlcNAc as substrates in in vitro reaction.The product GlcNAc-1-P was then separated by organic method using a sialic gel column.Lack of GlcNAc-1-P could be settled using this synthsis pathway in GlmU enzymatic study.
To further study on nucleotide triphosphate substrate specificity of the E.coli K12 GlmU-Tr229,site-directed mutaions were done in residues Gln76 and Gly81 of GlmU-Tr229. Biochemical characters of one of the mutations GlmU-Tr229 Q76E was well studied using UTP and CTP as nucleotide triphosphate,respectively.Capillary electrophoresis profiles showed that GlmU-Tr229 Q76E had a notable change in nucleotide triphosphate substrate specificity.CTP could be recognized and used in the formation of CDP-GlcNAc with a yield above 50%.However, the conversion ratio for UTP reactions was decreased to 50%in contrast with 95%at mixmum.in GlmU reaction.This work is still ongoing.
In sumary,this work provided a feasible approach for the synthesis of UDP-GlcNAc analogs which can be used to study GlcNAc-transferase reaction and various biosynthesis pathways for cell components.The systematic study on the broad substrate specificity of GlmU and GlmU-Tr229 from E.coli K12 could help us to enhance the basic knowledge of enzyme realted to amio-sugar nulceotide synthesis.
引文
(1) Sas, T. C, de Muinck Keizer-Schrama, S. M., Stijnen, T., Aanstoot, H. J., and Drop, S. L. (2000) Carbohydrate metabolism during long-term growth hormone (GH) treatment and after discontinuation of GH treatment in girls with Turner syndrome participating in a randomized dose-response study. Dutch Advisory Group on Growth Hormone. J Clin Endocrinol Metab 85, 769-75.
(2) Endo, T., Nishimura, R., Saito, S., Kanazawa, K., Nomura, K., Katsuno, M, Shii, K., Mukhopadhyay, K., Baba, S., and Kobata, A. (1992) Carbohydrate structures of beta-core fragments of human chorionic gonadotropin isolated from a pregnant individual. Endocrinology 131, 1457.
(3) Endo, T., Nishimura, R., Saito, S., Kanazawa, K., Nomura, K., Katsuno, M., Shii, K., Mukhopadhyay, K., Baba, S., and Kobata, A. (1992) Carbohydrate structures of beta-core fragment of human chorionic gonadotropin isolated from a pregnant individual. Endocrinology 130, 2052-8.
(4) Lucas, A. H., Apicella, M. A., and Taylor, C. E. (2005) Carbohydrate moieties as vaccine candidates. Clin Infect Dis 41, 705-12.
(5) Dabelsteen, E., Mandel, U., and Clausen, H. (1991) Cell surface carbohydrates are markers of differentiation in human oral epithelium. Crit Rev Oral Biol Med 2, 493-507.
(6) Gorelik, E., Galili, U., and Raz, A. (2001) On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis. Cancer Metastasis Rev 20, 245-77.
(7) Olden, K., Pratt, R. M, and Yamada, K. M. (1978) Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell 13, 461-73.
(8) McSweeney, L. A., and Dreyfus, L. A. (2005) Carbohydrate-binding specificity of the Escherichia coli cytolethal distending toxin CdtA-II and CdtC-II subunits. Infect Immun 73, 2051-60.
(9) Gornik,O.,Dumic,J.,Flogel,M.,and Lauc,G(2006) Glycoscience--a new frontier in rational drug design.Acta Pharm 56,19-30.
(10) Rademacher,T.W.,Parekh,R.B.,Dwek,R.A.,Isenberg,D.,Rook,G.,Axford,J.S.,and Roitt,I.(1988) The role of IgG glycoforms in the pathogenesis of rheumatoid arthritis.Springer Semin Immunopathol 10,231-49.
(11) Ratner,D.M.,Adams,E.W.,Disney,M.D.,and Seeberger,P.H.(2004) Tools for glycomics:mapping interactions of carbohydrates in biological systems.Chembiochem 5,1375-83.
(12) Gahmberg,C.G,and Tolvanen,M.(1996) Why mammalian cell surface proteins are glycoproteins.Trends Biochem Sci 21,308-11.
(13) Gilewski,T.,Ragupathi,G.,Bhuta,S.,Williams,L.J.,Musselli,C.,Zhang,X.F.,Bornmann,W.G.,Spassova,M.,Bencsath,K.P.,Panageas,K.S.,Chin,J.,Hudis,C.A.,Norton,L.,Houghton,A.N.,Livingston,P.O.,and Danishefsky,S.J.(2001) Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate:a phase Ⅰ trial.Proc Natl Acad Sci USA 98,3270-5.
(14) Wang,C.C.,Huang,Y.L.,Ren,C.T.,Lin,C.W.,Hung,J.T.,Yu,J.C.,Yu,A.L.,Wu,C.Y.,and Wong,C.H.(2008) Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer.Proc Natl Acad Sci USA 105,11661-6.
(15) 张树政(2002)糖生物学与糖生物工程,清华大学出版社,北京.
(16) Rademacher,T.W.,Parekh,R.B.,and Dwek,R.A.(1988) Glycobiology.Annu Rev Biochem 57,785-838.
(17) Alper,J.(2001) Searching for medicine's sweet spot.Science 291,2338-43.
(18) Brooks,S.A.,Carter,T.M.,Royle,L.,Harvey,D.J.,Fry,S.A.,Kinch,C.,Dwek,R.A.,and Rudd,P.M.(2008) Altered glycosylation of proteins in cancer:what is the potential for new anti-tumour strategies.Anticancer Agents Med Chem 8,2-21.
(19) Dube,D.H.,and Bertozzi,C.R.(2005) Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov 4,477-88.
(20) Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R. A. (2001) Glycosylation and the immune system. Science 291, 2370-6.
(21) Bertozzi, C. R., and Kiessling, L. L. (2001) Chemical glycobiology. Science 291, 2357-64.
(22) Helenius, A., and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science 291, 2364-9.
(23) Wells, L., Vosseller, K., and Hart, G W. (2001) Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 291, 2376-8.
(24) Nakahara, Y., Nonaka, T., Hojo, H., and Nakahara, Y. (2003) Synthesis of an unnatural N-glycan-linked dolichyl pyrophosphate precursor. Biosci Biotechnol Biochem 67, 1761-6.
(25) Koeller, K. M., and Wong, C. H. (2000) Complex carbohydrate synthesis tools for glycobiologists: enzyme-based approach and programmable one-pot strategies. Glycobiology 10, 1157-69.
(26) Burkhart, F., Zhang, Z., Wacowich-Sgarbi, S., and Wong, C. H. (2001) Synthesis of the Globo H Hexasaccharide Using the Programmable Reactivity-Based One-Pot Strategy This research was supported by the National Institutes of Health. F.B. thanks the Deutsche Forschungsgemeinschaft for a fellowship. Angew Chem Int Ed Engl 40, 1274-1277.
(27) Whitfield, C. (2006) Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem 75, 39-68.
(28) Guo, H., Yi, W., Song, J. K., and Wang, P. G. (2008) Current understanding on biosynthesis of microbial polysaeeharides. Curr Top Med Chem 8, 141-51.
(29) Joanne M.Williamson, M. S. A., Christian R.H.Raetz. (1991) Acyl-acyl carrier protein specificity of UDP-GlcNAc acetyltransferases from Gram-negative bacteria: Relationship to Lipid A structure. Journal of Bacteriology 173, 3591-3596.
(30) Raetz, C. R., and Whitfield, C. (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635-700.
(31) Jann, K., and Jann, B. (1992) Capsules of Escherichia coli, expression and biological significance. Can J Microbiol 38, 705-10.
(32) S.A.BarKer, A. B. F., M.Stacey J.M.Webber. (1958) Amino-sugars and related compounds.Part IV.Isolation and properties of oligosaccharides obtained by controlled fragmentation of chitin. J.Chem.Soc, 2218-2227.
(33) Ronge Xing, S. L., Huahua Yu, Zhaoyong Guo, Pibo Wang, Cuiping Li, Pengcheng Li. (2005) Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation. Carbohydr Res 340, 2150-2153.
(34) Se-Kwon Kim, N. R. (2005) Ensymatic production and biological activities of chitosan oligosaccharides(COS): A review. carbohydrate polymers 62, 357-368.
(35) Hu Zhan, Y. D., Xingju Yu, Masaru Mitsutomi, Sei-ichi Aiba. (1999) preparation of chitooligosaccharides from chitosan by a complex enzyme. Carbohydrate Research 320, 257-260.
(36) Prag, G., Papanikolau, Y., Tavlas, G., Vorgias, C. E., Petratos, K., and Oppenheim, A. B. (2000) Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540. J Mol Biol 300, 611-7.
(37) Tews, I., Dauter, Z., Oppenheim, A. B., and Vorgias, C. E. (1992) Crystallization of recombinant chitobiase from Serratia marcescens. J Mol Biol 228, 696-7.
(38) Kalabat, D. Y, Froelich, J. M., Phuong, T. K., Forsyth, R. A., Newman, V. G., and Zyskind, J. W. (1998) Chitobiase, a new reporter enzyme. Biotechniques 25, 1030-5.
(39) Lee, Y G., Chung, K. C., Wi, S. G., Lee, J. C., and Bae, H. J. (2009) Purification and properties of a chitinase from Penicillium sp. LYG 0704. Protein Expr Purif 65, 244-50.
(40) Keyhani, N. O., and Roseman, S. (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic beta-N-acetylglucosaminidase. J Biol Chem 271, 33425-32.
(41) Keyhani, N. O., and Roseman, S. (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic chitodextrinase. J Biol Chem 271, 33414-24.
(42) Keyhani, N. O., Wang, L. X., Lee, Y. C, and Roseman, S. (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Characterization of an N,N'-diacetyl-chitobiose transport system. J Biol Chem 271, 33409-13.
(43) Feng Tian, Y. L., Keao Hu, Binyuan Zhao. (2004) The depolymerization mechanism of chitosan by hydrogen peroxide. Journal of Materials Science 38, 4709-4712.
(44) Il'ina, A. V, Tkacheva Iu, V, and Varlamov, V. P. (2002) [Depolymerization of high-molecular-weight chitosan by the enzyme preparation Celloviridine G20x.]. Prikl Biokhim Mikrobiol 38, 132-5.
(45) Su, Y., Zhang,Houcheng, Gu,Li, Chen,Min, Xiao,Min,Wang,Peng George, Qi,Qingsheng. (2007) Engineered yeast with PNGaseF on cell surface for releasing of N-Glycans from glycoprotein. Enzyme and Microbial Technology 40, 1496-1502.
(46) Abe, H., Shimma, Y, and Jigami, Y. (2003) In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir. Glycobiology 13, 87-95.
(47) Perugino, G., Trincone, A., Rossi, M., and Moracci, M. (2004) Oligosaccharide synthesis by glycosynthases. Trends Biotechnol 22, 31-7.
(48) Bucke, C. (1999) Review oligosaccharide synthesis using glycosidases. Journal of Chemical Technology &Biotechnology 67, 217-220.
(49) Mushegian, J. L. A. (2003) Three monophyletic superfamilies account for the majority of the known glycosyltransferases. Protein Science 72,1418-1431.
(50) Leloir, L. F., De Fekete, M. A., and Cardini, C. E. (1961) Starch and oligosaccharide synthesis from uridine diphosphate glucose.J Biol Chem 236,636-41.
(51) Weijers,C.A.,Franssen,M.C.,and Visser,G.M.(2008)Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides.Biotechnol Adv 26,436-56.
(52) Rich,J.R.,Szpacenko,A.,Palcic,M.M.,and Bundle,D.R.(2004)Glycosyltransferase-catalyzed synthesis of thiooligosaccharides.Angew Chem Int Ed Eng143,613-5.
(53) Koeller,K.M.,Smith,M.E.,and Wong,C.H.(2000) Chemoenzymatic synthesis of PSGL-1 glycopeptides:sulfation on tyrosine affects glycosyltransferase-catalyzed synthesis of the O-glycan.Bioorg Med Chem 8,1017-25.
(54) Chen,X.,Liu,Z.,Zhang,J.,Zhang,W.,Kowal,P.,and Wang,P.G.(2002)Reassembled biosynthetic pathway for large-scale carbohydrate synthesis:alpha-Gal epitope producing "superbug".Chembiochem 3,47-53.
(55) Shao,J.,Zhang,J.,Kowal,P.,Lu,Y.,and Wang,P.G.(2002) Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd.Biochem Biophys Res Commun 295,1-8.
(56) Zhang,J.,Chen,X.,Shao,J.,Liu,Z.,Kowal,P.,Lu,Y.,and Wang,P.G.(2003)Synthesis of galactose-containing oligosaccharides through superbeads and superbug approaches:substrate recognition along different biosynthetic pathways.Methods Enzymol 362,106-24.
(57) Ravindranath,R.M.,and Graves,M.C.(1990) Attenuated murine cytomegalovirus binds to N-acetylglucosamine,and shift to virulence may involve recognition of sialic acids.J Virol 64,5430-40.
(58) Thompson,J.N.,Jones,M.Z.,Dawson,G.,and Huffman,P.S.(1992)N-acetylglucosamine 6-sulphatase deficiency in a Nubian goat:a model of Sanfilippo syndrome type D(mucopolysaccharidosis ⅢD).J Inherit Metab Dis 15,760-8.
(59) Jaeken, J. (1989) [A not-previously described hereditary neurological disease with a deficiency of sialic acid, galactose and N-acetylglucosamine of plasma glycoproteins]. Verh K Acad Geneeskd Belg 51, 377-406.
(60) Gaily, D., Bray, K., and Cooper, S. (1993) Synthesis of peptidoglycan and membrane during the division cycle of rod-shaped, gram-negative bacteria. J Bacteriol 175,3121-30.
(61) Stoolmiller, A. C, and Dorfman, A. (1969) The biosynthesis of hyaluronic acid by Streptococcus. J Biol Chem 244, 236-46.
(62) Lansing, M., Lellig, S., Mausolf, A., Martini, I., Crescenzi, F., O'Regan, M, and Prehm, P. (1993) Hyaluronate synthase: cloning and sequencing of the gene from Streptococcus sp. Biochem J 289 (Pt 1), 179-84.
(63) Nagarajan, M., and Rao, V. S. (1979) Conformational studies of proteoglycans: theoretical studies on the conformation of heparin. Biopolymers 18, 1407-20.
(64) Lee, J. C, Lu, X. A., Kulkarni, S. S, Wen, Y. S., and Hung, S. C. (2004) Synthesis of heparin oligosaccharides. J Am Chem Soc 126, 476-7.
(65) Zhang, H., Muramatsu, T., Murase, A., Yuasa, S., Uchimura, K., and Kadomatsu, K. (2006) N-Acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury. Glycobiology 16, 702-10.
(66) Funderburgh, J. L. (2000) Keratan sulfate: structure, biosynthesis, and function. Glycobiology 10, 951-8.
(67) Ginsburg, V. (1978) Comparative biochemistry of nucleotide-linked sugars. Prog Clin Biol Res 23, 595-600.
(68) Plumbridge, J. A., Cochet, O., Souza, J. M., Altamirano, M. M., Calcagno, M. L., and Badet, B. (1993) Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12. J Bacteriol 175, 4951-6.
(69) Knop, J. K., and Hansen, R. G. (1970) Uridine diphosphate glucose pyrophosphorylase. IV. Crystallization and properties of the enzyme from human liver. J Biol Chem 245, 2499-504.
(70) Bulter, T., and Elling, L. (1999) Enzymatic synthesis of nucleotide sugars. Glycoconj J 16, 147-59.
(71) Cabib, E., Carminatti, H., and Woyskovsky, N. M. (1965) Phosphorolysis Of The Pyrophosphate Bond Of Sugar Nucleotides. Ii. Purification And Properties Of The Enzyme. J Biol Chem 240, 2114-21.
(72) Carminatti, H., and Cabib, E. (1965) Phosphorolysis Of The Pyrophosphate Bond Of Sugar Nucleotides. I. Characterization And Stoichiometry Of The Reaction. J Biol Chem 240,2110-3.
(73) Uehara, T., and Park, J. T. (2004) The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling. J Bacteriol 186, 7273-9.
(74) Lunin, V. V, Li, Y., Schrag, J. D., Iannuzzi, P., Cygler, M., and Matte, A. (2004) Crystal structures of Escherichia coli ATP-dependent glucokinase and its complex with glucose. J Bacteriol 186, 6915-27.
(75) Meyer, D., Schneider-Fresenius, C., Horlacher, R., Peist, R., and Boos, W. (1997) Molecular characterization of glucokinase from Escherichia coli K-12. J Bacteriol 179, 1298-306.
(76) Hofmann, M., Boles, E., and Zimmermann, F. K. (1994) Characterization of the essential yeast gene encoding N-acetylglucosamine-phosphate mutase. Eur J Biochem 221,741-7.
(77) Jolly, L., Ferrari, P., Blanot, D., Van Heijenoort, J., Fassy, F., and Mengin-Lecreulx, D. (1999) Reaction mechanism of phosphoglucosamine mutase from Escherichia coli. Eur J Biochem 262, 202-10.
(78) Mao, Z., Shin, H. D., and Chen, R. R. (2006) Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis. Biotechnol Prog 22,369-74.
(79) Abeijon, C., Robbins, P. W., and Hirschberg, C. B. (1996) Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis. Proc Natl Acad Sci U S A 93, 5963-8.
(80) Martinez-Duncker, I., Mollicone, R., Codogno, P., and Oriol, R. (2003) The nucleotide-sugar transporter family: a phylogenetic approach. Biochimie 85, 245-60.
(81) Berninsone, P., Miret, J. J., and Hirschberg, C. B. (1994) The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles. J Biol Chem 269, 207-11.
(82) Hirschberg, C. B., Robbins, P. W., and Abeijon, C. (1998) Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 67, 49-69.
(83) Kawakita, M., Ishida, N., Miura, N., Sun-Wada, G. H., and Yoshioka, S. (1998) Nucleotide sugar transporters: elucidation of their molecular identity and its implication for future studies. J Biochem 123, 777-85.
(84) Gerardy-Schahn, R., Oelmann, S., and Bakker, H. (2001) Nucleotide sugar transporters: biological and functional aspects. Biochimie 83, 775-82.
(85) Berninsone, P., and Hirschberg, C. B. (1998) Nucleotide sugars, nucleotide sulfate, and ATP transporters of the endoplasmic reticulum and Golgi apparatus. Ann N Y Acad Sci 842, 91-9.
(86) Yamamoto, K., Kawai, H., and Tochikura, T. (1981) Preparation of Uridine Diphosphate-N-Acetylgalactosamine from Uridine Diphosphate-N-Acetylglucosamine by Using Microbial Enzymes. Appl Environ Micwbiol 41, 392-395.
(87) Allard, S. T., Giraud, M. F., and Naismith, J. H. (2001) Epimerases: structure, function and mechanism. Cell Mol Life Sci 58, 1650-65.
(88) Dormann, P., and Benning, C. (1998) The role of UDP-glucose epimerase in carbohydrate metabolism of Arabidopsis. Plant J13, 641-52.
(89) Lopez, A. B., Sener, K., Trosien, J., Jarroll, E. L., and van Keulen, H. (2007) UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity. J Eukaryot Micwbiol 54, 154-60.
(90) Guo, H., Li, L., and Wang, P. G (2006) Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7. Biochemistry 45, 13760-8.
(91) Schulz, J. M., Watson, A. L., Sanders, R., Ross, K. L., Thoden, J. B., Holden, H.M.,and Fridovich-Keil,J.L.(2004) Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase.J Biol Chem 279,32796-803.
(92) Thoden JB,W.T.,Fridovich-Keil JL,Holden HM.(2000) Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase.Biochemistry 39,5691-5701.
(93) Guo,H.,Yi,W.,Li,L.,and Wang,P.G.(2007) Three UDP-hexose 4-epimerases with overlapping substrate specificity coexist in E.coli O86:B7.Biochem Biophys Res Commun 356,604-9.
(94) Chen,X.,Fang,J.,Zhang,J.,Liu,Z.,Shao,J.,Kowal,P.,Andreana,P.,and Wang,P.G.(2001) Sugar nucleotide regeneration beads(superbeads):a versatile tool for the practical synthesis of oligosaccharides.J Am Chem Soc 123,2081-2.
(95) Fang,J.,Chen,X.,Zhang,W.,Janczuk,A.,and Wang,P.G.(2000) Synthesis of alpha-Gal epitope derivatives with a galactosyltransferase-epimerase fusion enzyme.Carbohydr Res 329,873-8.
(96) Wu B,Z.Y.,Zheng R,Guo C,Wang PG.(2002) Bifunctional phosphomannose isomerase/GDP-D-mannose pyrophosphorylase is the point of control for GDP-D-mannose biosynthesis in Helicobacter pylori.FEBS Lett 519,87-92.
(97) Wu B,Z.Y.,Wang PG.(2001) Identification and characterization of GDP-d-mannose 4,6-dehydratase and GDP-1-fucose snthetase in a GDP-1-fucose biosynthetic gene cluster from Helicobacter pylori.Biochem Biophys Res Commun.285,364-371.
(98) Nakagawa,J.,Tamaki,S.,Tomioka,S.,and Matsuhashi,M.(1984) Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides.Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase.J Biol Chem 259,13937-46.
(99) Williamson,J.M.,Anderson,M.S.,and Raetz,C.R.(1991) Acyl-acyl carrier protein specificity ofUDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure. J Bacteriol 173, 3591-6.
(100) Kuhn, H. M., Meier-Dieter, U., and Mayer, H. (1988) ECA, the enterobacterial common antigen. FEMS Microbiol Rev 4,195-222.
(101) Chang, R., Moquist, P., and Finney, N. S. (2004) Chemical synthesis of UDP-4-O-methyl-GlcNAc, a potential chain terminator of chitin synthesis. Carbohydr Res 339,1531-6.
(102) Bulik, D. A., Olczak, M., Lucero, H. A., Osmond, B. C, Robbins, P. W., and Specht, C. A. (2003) Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress. Eukaryot Cell 2, 886-900.
(103) Herscovics, A., and Orlean, P. (1993) Glycoprotein biosynthesis in yeast. Faseb J 7, 540-50.
(104) Namboori, S. C., and Graham, D. E. (2008) Acetamido sugar biosynthesis in the Euryarchaea. J Bacteriol 190, 2987-96.
(105) Levy, P., and Picard, J. (1976) Glycosaminoglycan biosynthesis in arterial wall. Hexosaminyltransferase and glucuronyltransferase in cell membranes of aortic media-intima. Eur J Biochem 61, 613-9.
(106) Feng, F., Okuyama, K., Niikura, K., Ohta, T., Sadamoto, R., Monde, K., Noguchi, T., and Nishimura, S. (2004) Chemo-enzymatic synthesis of fluorinated 2-N-acetamidosugar nucleotides using UDP-GlcNAc pyrophosphorylase. Org Biomol Chem 2, 1617-23.
(107) Cecchelli, R., Cacan, R., and Verbert, A. (1985) Accumulation of UDP-GlcNAc into intracellular vesicles and occurrence of a carrier-mediated transport. Study with plasma-membrane-permeabilized mouse thymocytes. Eur J Biochem 153, 111-6.
(108) Sasai, K., Ikeda, Y., Fujii, T., Tsuda, T., and Taniguchi, N. (2002) UDP-GlcNAc concentration is an important factor in the biosynthesis of betal,6-branched oligosaccharides: regulation based on the kinetic properties of N-acetylglucosaminyltransferase V. Glycobiology 12, 119-27.
(109) Boehmelt, G, Wakeham, A., Elia, A., Sasaki, T., Plyte, S., Potter, J., Yang, Y., Tsang,E.,Ruland,J.,Iscove,N.N.,Dennis,J.W.,and Mak,T.W.(2000)Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells.Embo J 19,5092-104.
(110) Stokes,M.J.,Guther,M.L.,Turnock,D.C.,Prescott,A.R.,Martin,K.L.,Alphey,M.S.,and Ferguson,M.A.(2008) The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation.J Biol Chem 283,16147-61.
(111) Mio,T.,Yabe,T.,Arisawa,M.,and Yamada-Okabe,H.(1998) The eukaryotic UDP-N-acetylglucosamine pyrophosphorylases.Gene cloning,protein expression,and catalytic mechanism.J Biol Chem 273,14392-7.
(112) Milewski,S.,Gabriel,I.,and Olchowy,J.(2006) Enzymes of UDP-GlcNAc biosynthesis in yeast.Yeast 23,1-14.
(113) m.Huang,L.M.G.(2002)De novo synthesis of pyrimidine nucleotides;emerging interfaces with signal transduction pathways.Cell.Mol.Life Sci.60,321-336.
(114) Yamada-Okabe,T.,Sakamori,Y.,Mio,Y.,and Yamada-Okabe,H.(2001)Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.Eur J Biochem 268,2498-505.
(115) Shepherd,J.G.,Wang,L.,and Reeves,P.R.(2000) Comparison of O-antigen gene clusters of Escherichia coli(Shigella) sonnei and Plesiomonas shigelloides O17:sonnei gained its current plasmid-borne O-antigen genes from P.shigelloides in a recent event.Infect Immun 68,6056-61.
(116) Okuyama,K.,Hamamoto,T.,Ishige,K.,Takenouchi,K.,and Noguchi,T.(2000) An efficient method for production of uridine 5'-diphospho-N-acetylglucosamine.Biosci Biotechnol Biochem 64,386-92.
(117) Chang,R.,Vo,T.T.,and Finney,N.S.(2006) Synthesis of the C1-phosphonate analog of UDP-GlcNAc.Carbohydr Res 341,1998-2004.
(118) Schafer,A.,and Thiem,J.(2000) Synthesis of novel donor mimetics of UDP-Gal,UDP-GlcNAc,and UDP-GalNAc as potential transferase inhibitors.J Org Chem 65,24-9.
(119) Jurgen E.Heidlas,W.J.L.,Patrick Pale,George M.Whitesides.(1992)Gram-scales synthesis of Uridine 5'-Diphospho-N-acetylglucosamine:Comparison of Enzymatic and Chemical Routes.J.Org.Chem 57,146-151.
(120) Jungdon Bae,K.-H.K.,Dooil Kim,Yongseok Choi,Joong Su Kim,Sukhoon Hoh,Suk-In Hong,Dae-Sil Lee.(2005) A practical enzymatic synthesis of UDP-sugars and NDP Glucoses.Chembiochem 6,1963-1966.
(121) Leiting,B.,Pryor,K.D.,Eveland,S.S.,and Anderson,M.S.(1998) One-day enzymatic synthesis and purification of UDP-N-[1-14C]acetyl-glucosamine.Anal Biochem 256,185-91.
(122) Tochikura,Y.K.,H.,Gotan,T.(1971) studies on microbial metabolism of sugar nucleotides.Part Ⅵ.Agric.Biol.Chem.35,163-176.
(123) Jun Shao,J.Z.,Jozef Nahalka Peng George Wang.(2002) Biocatalytic synthesis of uridine 5'-diphosphate N-acetylglucosamine by multiple enzymes co-immobilized on agarose beads.Chem.Commun.,2586-2587.
(124) Koizumi,S.,Endo,T.,Tabata,K.,and Ozaki,A.(1998) Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria.Nat Biotechnol 16,847-50.
(125) Kazuhibo Tabata,S.K.,Tetsuo Endo,Akio Ozaki.(2000) Production of UDP-N-acetylglucosamine by coupling metabolically engineered bacteria.Biotechnology Letters 22,479-483.
(126) Gary C.Look,C.H.F.,Chi Huey Wong.(1993) Enzyme-catalyzed organic synthesis:practical routes to aza sugars and their analogs for use as glycprocessing inhibitors.Acc.Chem.Res.26,181-190.
(127) Sunthankar,P.,Pastuszak,I.,Rooke,A.,Elbein,A.D.,van de Rijn,I.,Canfield,W.M.,and Drake,R.R.(1998) Synthesis of 5-azido-UDP-N-acetylhexosamine photoaffinity analogs and radiolabeled UDP-N-acetylhexosamines.Anal Biochem 258,195-201.
(128) Pinney, K. G., Mejia, M. P., Villalobos, V. M., Rosenquist, B. E., Pettit, G R., Verdier-Pinard, P., and Hamel, E. (2000) Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin. Bioorg Med Chem 8, 2417-25.
(129) Jurgen E. Heidlas, W. J. L., Patrick Pale, George M. Whitesides. (1992) Gram-scale synthesis of uridine 5'-diphospho-N-acetylglucosamine: comparison of enzymic and chemical routes. J.Org.Chem 57,146-151.
(130) Togayachi, A., Akashima, T., Ookubo, R., Kudo, T., Nishihara, S., Iwasaki, H., Natsume, A., Mio, H., Inokuchi, J., Irimura, T., Sasaki, K., and Narimatsu, H. (2001) Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids. J Biol Chem 276, 22032-40.
(131) Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteornics 8, 3294-302.
(132) Kamst, E., Pilling, J., Raamsdonk, L. M., Lugtenberg, B. J., and Spaink, H. P. (1997) Rhizobium nodulation protein NodC is an important determinant of chitin oligosaccharide chain length in Nod factor biosynthesis. J Bacteriol 179, 2103-8.
(133) Chen, W. Y., Marcellin, E., Hung, J., and Nielsen, L. K. (2009) Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus. J Biol Chem 284,18007-14.
(134) Lee, F. K., Stephens, D. S., Gibson, B. W., Engstrom, J. J., Zhou, D., and Apicella, M. A. (1995) Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB. Infect Immun 63, 2508-15.
(135) Hanover, J. A. (2001) Glycan-dependent signaling: O-linked N-acetylglucosamine. Faseb J 15,1865-76.
(136) Pattabiraman, T. N., and Bachhawat, B. K. (1961) Purification of uridine diphosphoacetylglucosamine pyrophosphorylase from sheep brain. Biochim Biophys Acta 50, 129-34.
(137) Szumilo, T., Zeng, Y, Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) Purification to homogeneity and properties of UDP-GlcNAc (GalNAc) pyrophosphorylase. J Biol Chem 271, 13147-54.
(138) Maruyama, D., Nishitani, Y., Nonaka, T., Kita, A., Fukami, T. A., Mio, T., Yamada-Okabe, H., Yamada-Okabe, T., and Miki, K. (2007) Crystal structure of uridine-diphospho-N-acetylglucosamine pyrophosphorylase from Candida albicans and catalytic reaction mechanism. J Biol Chem 282,17221-30.
(139) Bulik, D. A., van Ophem, P., Manning, J. M., Shen, Z., Newburg, D. S., and Jarroll, E. L. (2000) UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated. J Biol Chem 275, 14722-8.
(140) Mengin-Lecreulx, D., and van Heijenoort, J. (1993) Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli. J Bacteriol 175, 6150-7.
(141) Pompeo, F., Bourne, Y, van Heijenoort, J., Fassy, F., and Mengin-Lecreulx, D. (2001) Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth. J Biol Chem 276, 3833-9.
(142) Mengin-Lecreulx, D., and van Heijenoort, J. (1994) Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis. J Bacteriol 176, 5788-95.
(143) Mochalkin, I., Lightle, S., Zhu, Y, Ohren, J. F., Spessard, C., Chirgadze, N. Y., Banotai, C., Melnick, M., and McDowell, L. (2007) Characterization of substrate binding and catalysis in the potential antibacterial target N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). Protein Sci 16, 2657-66.
(144) Olsen, L. R., and Roderick, S. L. (2001) Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites. Biochemistry 40, 1913-21.
(145) Gehring, A. M., Lees, W. J., Mindiola, D. J., Walsh, C. T., and Brown, E. D. (1996) Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli. Biochemistry 35, 579-85.
(146) Parisi, G, Fornasari, M. S., and Echave, J. (2004) Dynactins p25 and p27 are predicted to adopt the LbetaH fold. FEBS Lett 562, 1-4.
(147) Parisi, G, and Echave, J. (2004) The structurally constrained protein evolution model accounts for sequence patterns of the LbetaH superfamily. BMC Evol Biol 4,41.
(148) Pompeo, F., van Heijenoort, J., and Mengin-Lecreulx, D. (1998) Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes. J Bacteriol 180, 4799-803.
(149) Olsen, L. R., Vetting, M. W., and Roderick, S. L. (2007) Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products. Protein Sci 16, 1230-5.
(150) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72,248-54.
(151) Zenker, A., Galanski, M., Bereuter, T. L., Keppler, B. K., and Lindner, W. (1999) Capillary electrophoretic study of cisplatin interaction with nucleoside monophosphates, di- and trinucleotides. J Chromatogr A 852, 337-46.
(152) Kostrewa, D., D'Arcy, A., Takacs, B., and Kamber, M. (2001) Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase,GlmU,in apo form at 2.33 A resolution and in complex with UDP-N-acetylglucosamine and Mg(2+) at 1.96 A resolution.J Mol Biol 305,279-89.
(153) Evans,C.E.,and Lovell,P.A.(2009) Click chemistry as a route to surface functionalization of polymer particles dispersed in aqueous media.Chem Commun(Camb),2305-7.
(154) Leavy,T.M.,and Bertozzi,C.R.(2007) A high-throughput assay for O-GlcNAc transferase detects primary sequence preferences in peptide substrates.Bioorg Med Chem Lett 17,3851-4.
(155) Hang,H.C.,Yu,C.,Kato,D.L.,and Bertozzi,C.R.(2003) Ametabolic labeling approach toward proteomic analysis ofmucin-type O-linked glycosylation.Proc Natl A cad Sci U S A 100,14846-51.
(156) Bond,M.R.,and Kohler,J.J.(2007) Chemical methods for glycoprotein discovery.Curr Opin Chem Biol 11,52-8.
(157) Vocadlo,D.J.,Hang,H.C.,Kim,E.J.,Hanover,J.A.,and Bertozzi,C.R.(2003) A chemical approach for identifying O-GlcNAc-modified proteins in cells.Proc Natl Acad Sci USA 100,9116-21.
(158) Guisheng Zhang,L.F.,Lizhi Zhu,Yanqiang Zhong,Peng George Wang,Duxin Sun.(2006) syntheses and biological activities of 3'-azido disaccharide analogues of daunorubicin against drug-resistant leukemia.J.Med.Chem.29,1792-1799.
(159) Saxon,E.,Luchansky,S.J.,Hang,H.C.,Yu,C.,Lee,S.C.,and Bertozzi,C.R.(2002) Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation.J Am Chem Soc 124,14893-902.
(160) Ruhela,D.,and Vishwakarma,R.A.(2003) Iterative synthesis of Leishmania phosphoglycans by solution,solid-phase,and polycondensation approaches without involving any glycosylation.J Org Chem 68,4446-56.
(161) Sven Fey,L.E.,Udo Kragl.(1997) The cofactor Mg2+--a key switch for effective continuous enzymatic production of GDP-mannose using recombinant GDP-mannose pyrophosphorylase.Carbohydrate Research 305, 475-481.
(162) Nishimoto,M.,and Kitaoka,M.(2007) Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum.Appl Environ Microbiol 73,6444-9.
(2) Endo, T., Nishimura, R., Saito, S., Kanazawa, K., Nomura, K., Katsuno, M, Shii, K., Mukhopadhyay, K., Baba, S., and Kobata, A. (1992) Carbohydrate structures of beta-core fragments of human chorionic gonadotropin isolated from a pregnant individual. Endocrinology 131, 1457.
(3) Endo, T., Nishimura, R., Saito, S., Kanazawa, K., Nomura, K., Katsuno, M., Shii, K., Mukhopadhyay, K., Baba, S., and Kobata, A. (1992) Carbohydrate structures of beta-core fragment of human chorionic gonadotropin isolated from a pregnant individual. Endocrinology 130, 2052-8.
(4) Lucas, A. H., Apicella, M. A., and Taylor, C. E. (2005) Carbohydrate moieties as vaccine candidates. Clin Infect Dis 41, 705-12.
(5) Dabelsteen, E., Mandel, U., and Clausen, H. (1991) Cell surface carbohydrates are markers of differentiation in human oral epithelium. Crit Rev Oral Biol Med 2, 493-507.
(6) Gorelik, E., Galili, U., and Raz, A. (2001) On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis. Cancer Metastasis Rev 20, 245-77.
(7) Olden, K., Pratt, R. M, and Yamada, K. M. (1978) Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell 13, 461-73.
(8) McSweeney, L. A., and Dreyfus, L. A. (2005) Carbohydrate-binding specificity of the Escherichia coli cytolethal distending toxin CdtA-II and CdtC-II subunits. Infect Immun 73, 2051-60.
(9) Gornik,O.,Dumic,J.,Flogel,M.,and Lauc,G(2006) Glycoscience--a new frontier in rational drug design.Acta Pharm 56,19-30.
(10) Rademacher,T.W.,Parekh,R.B.,Dwek,R.A.,Isenberg,D.,Rook,G.,Axford,J.S.,and Roitt,I.(1988) The role of IgG glycoforms in the pathogenesis of rheumatoid arthritis.Springer Semin Immunopathol 10,231-49.
(11) Ratner,D.M.,Adams,E.W.,Disney,M.D.,and Seeberger,P.H.(2004) Tools for glycomics:mapping interactions of carbohydrates in biological systems.Chembiochem 5,1375-83.
(12) Gahmberg,C.G,and Tolvanen,M.(1996) Why mammalian cell surface proteins are glycoproteins.Trends Biochem Sci 21,308-11.
(13) Gilewski,T.,Ragupathi,G.,Bhuta,S.,Williams,L.J.,Musselli,C.,Zhang,X.F.,Bornmann,W.G.,Spassova,M.,Bencsath,K.P.,Panageas,K.S.,Chin,J.,Hudis,C.A.,Norton,L.,Houghton,A.N.,Livingston,P.O.,and Danishefsky,S.J.(2001) Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate:a phase Ⅰ trial.Proc Natl Acad Sci USA 98,3270-5.
(14) Wang,C.C.,Huang,Y.L.,Ren,C.T.,Lin,C.W.,Hung,J.T.,Yu,J.C.,Yu,A.L.,Wu,C.Y.,and Wong,C.H.(2008) Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer.Proc Natl Acad Sci USA 105,11661-6.
(15) 张树政(2002)糖生物学与糖生物工程,清华大学出版社,北京.
(16) Rademacher,T.W.,Parekh,R.B.,and Dwek,R.A.(1988) Glycobiology.Annu Rev Biochem 57,785-838.
(17) Alper,J.(2001) Searching for medicine's sweet spot.Science 291,2338-43.
(18) Brooks,S.A.,Carter,T.M.,Royle,L.,Harvey,D.J.,Fry,S.A.,Kinch,C.,Dwek,R.A.,and Rudd,P.M.(2008) Altered glycosylation of proteins in cancer:what is the potential for new anti-tumour strategies.Anticancer Agents Med Chem 8,2-21.
(19) Dube,D.H.,and Bertozzi,C.R.(2005) Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov 4,477-88.
(20) Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., and Dwek, R. A. (2001) Glycosylation and the immune system. Science 291, 2370-6.
(21) Bertozzi, C. R., and Kiessling, L. L. (2001) Chemical glycobiology. Science 291, 2357-64.
(22) Helenius, A., and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science 291, 2364-9.
(23) Wells, L., Vosseller, K., and Hart, G W. (2001) Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 291, 2376-8.
(24) Nakahara, Y., Nonaka, T., Hojo, H., and Nakahara, Y. (2003) Synthesis of an unnatural N-glycan-linked dolichyl pyrophosphate precursor. Biosci Biotechnol Biochem 67, 1761-6.
(25) Koeller, K. M., and Wong, C. H. (2000) Complex carbohydrate synthesis tools for glycobiologists: enzyme-based approach and programmable one-pot strategies. Glycobiology 10, 1157-69.
(26) Burkhart, F., Zhang, Z., Wacowich-Sgarbi, S., and Wong, C. H. (2001) Synthesis of the Globo H Hexasaccharide Using the Programmable Reactivity-Based One-Pot Strategy This research was supported by the National Institutes of Health. F.B. thanks the Deutsche Forschungsgemeinschaft for a fellowship. Angew Chem Int Ed Engl 40, 1274-1277.
(27) Whitfield, C. (2006) Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem 75, 39-68.
(28) Guo, H., Yi, W., Song, J. K., and Wang, P. G. (2008) Current understanding on biosynthesis of microbial polysaeeharides. Curr Top Med Chem 8, 141-51.
(29) Joanne M.Williamson, M. S. A., Christian R.H.Raetz. (1991) Acyl-acyl carrier protein specificity of UDP-GlcNAc acetyltransferases from Gram-negative bacteria: Relationship to Lipid A structure. Journal of Bacteriology 173, 3591-3596.
(30) Raetz, C. R., and Whitfield, C. (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635-700.
(31) Jann, K., and Jann, B. (1992) Capsules of Escherichia coli, expression and biological significance. Can J Microbiol 38, 705-10.
(32) S.A.BarKer, A. B. F., M.Stacey J.M.Webber. (1958) Amino-sugars and related compounds.Part IV.Isolation and properties of oligosaccharides obtained by controlled fragmentation of chitin. J.Chem.Soc, 2218-2227.
(33) Ronge Xing, S. L., Huahua Yu, Zhaoyong Guo, Pibo Wang, Cuiping Li, Pengcheng Li. (2005) Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation. Carbohydr Res 340, 2150-2153.
(34) Se-Kwon Kim, N. R. (2005) Ensymatic production and biological activities of chitosan oligosaccharides(COS): A review. carbohydrate polymers 62, 357-368.
(35) Hu Zhan, Y. D., Xingju Yu, Masaru Mitsutomi, Sei-ichi Aiba. (1999) preparation of chitooligosaccharides from chitosan by a complex enzyme. Carbohydrate Research 320, 257-260.
(36) Prag, G., Papanikolau, Y., Tavlas, G., Vorgias, C. E., Petratos, K., and Oppenheim, A. B. (2000) Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540. J Mol Biol 300, 611-7.
(37) Tews, I., Dauter, Z., Oppenheim, A. B., and Vorgias, C. E. (1992) Crystallization of recombinant chitobiase from Serratia marcescens. J Mol Biol 228, 696-7.
(38) Kalabat, D. Y, Froelich, J. M., Phuong, T. K., Forsyth, R. A., Newman, V. G., and Zyskind, J. W. (1998) Chitobiase, a new reporter enzyme. Biotechniques 25, 1030-5.
(39) Lee, Y G., Chung, K. C., Wi, S. G., Lee, J. C., and Bae, H. J. (2009) Purification and properties of a chitinase from Penicillium sp. LYG 0704. Protein Expr Purif 65, 244-50.
(40) Keyhani, N. O., and Roseman, S. (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic beta-N-acetylglucosaminidase. J Biol Chem 271, 33425-32.
(41) Keyhani, N. O., and Roseman, S. (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of a periplasmic chitodextrinase. J Biol Chem 271, 33414-24.
(42) Keyhani, N. O., Wang, L. X., Lee, Y. C, and Roseman, S. (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Characterization of an N,N'-diacetyl-chitobiose transport system. J Biol Chem 271, 33409-13.
(43) Feng Tian, Y. L., Keao Hu, Binyuan Zhao. (2004) The depolymerization mechanism of chitosan by hydrogen peroxide. Journal of Materials Science 38, 4709-4712.
(44) Il'ina, A. V, Tkacheva Iu, V, and Varlamov, V. P. (2002) [Depolymerization of high-molecular-weight chitosan by the enzyme preparation Celloviridine G20x.]. Prikl Biokhim Mikrobiol 38, 132-5.
(45) Su, Y., Zhang,Houcheng, Gu,Li, Chen,Min, Xiao,Min,Wang,Peng George, Qi,Qingsheng. (2007) Engineered yeast with PNGaseF on cell surface for releasing of N-Glycans from glycoprotein. Enzyme and Microbial Technology 40, 1496-1502.
(46) Abe, H., Shimma, Y, and Jigami, Y. (2003) In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir. Glycobiology 13, 87-95.
(47) Perugino, G., Trincone, A., Rossi, M., and Moracci, M. (2004) Oligosaccharide synthesis by glycosynthases. Trends Biotechnol 22, 31-7.
(48) Bucke, C. (1999) Review oligosaccharide synthesis using glycosidases. Journal of Chemical Technology &Biotechnology 67, 217-220.
(49) Mushegian, J. L. A. (2003) Three monophyletic superfamilies account for the majority of the known glycosyltransferases. Protein Science 72,1418-1431.
(50) Leloir, L. F., De Fekete, M. A., and Cardini, C. E. (1961) Starch and oligosaccharide synthesis from uridine diphosphate glucose.J Biol Chem 236,636-41.
(51) Weijers,C.A.,Franssen,M.C.,and Visser,G.M.(2008)Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides.Biotechnol Adv 26,436-56.
(52) Rich,J.R.,Szpacenko,A.,Palcic,M.M.,and Bundle,D.R.(2004)Glycosyltransferase-catalyzed synthesis of thiooligosaccharides.Angew Chem Int Ed Eng143,613-5.
(53) Koeller,K.M.,Smith,M.E.,and Wong,C.H.(2000) Chemoenzymatic synthesis of PSGL-1 glycopeptides:sulfation on tyrosine affects glycosyltransferase-catalyzed synthesis of the O-glycan.Bioorg Med Chem 8,1017-25.
(54) Chen,X.,Liu,Z.,Zhang,J.,Zhang,W.,Kowal,P.,and Wang,P.G.(2002)Reassembled biosynthetic pathway for large-scale carbohydrate synthesis:alpha-Gal epitope producing "superbug".Chembiochem 3,47-53.
(55) Shao,J.,Zhang,J.,Kowal,P.,Lu,Y.,and Wang,P.G.(2002) Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd.Biochem Biophys Res Commun 295,1-8.
(56) Zhang,J.,Chen,X.,Shao,J.,Liu,Z.,Kowal,P.,Lu,Y.,and Wang,P.G.(2003)Synthesis of galactose-containing oligosaccharides through superbeads and superbug approaches:substrate recognition along different biosynthetic pathways.Methods Enzymol 362,106-24.
(57) Ravindranath,R.M.,and Graves,M.C.(1990) Attenuated murine cytomegalovirus binds to N-acetylglucosamine,and shift to virulence may involve recognition of sialic acids.J Virol 64,5430-40.
(58) Thompson,J.N.,Jones,M.Z.,Dawson,G.,and Huffman,P.S.(1992)N-acetylglucosamine 6-sulphatase deficiency in a Nubian goat:a model of Sanfilippo syndrome type D(mucopolysaccharidosis ⅢD).J Inherit Metab Dis 15,760-8.
(59) Jaeken, J. (1989) [A not-previously described hereditary neurological disease with a deficiency of sialic acid, galactose and N-acetylglucosamine of plasma glycoproteins]. Verh K Acad Geneeskd Belg 51, 377-406.
(60) Gaily, D., Bray, K., and Cooper, S. (1993) Synthesis of peptidoglycan and membrane during the division cycle of rod-shaped, gram-negative bacteria. J Bacteriol 175,3121-30.
(61) Stoolmiller, A. C, and Dorfman, A. (1969) The biosynthesis of hyaluronic acid by Streptococcus. J Biol Chem 244, 236-46.
(62) Lansing, M., Lellig, S., Mausolf, A., Martini, I., Crescenzi, F., O'Regan, M, and Prehm, P. (1993) Hyaluronate synthase: cloning and sequencing of the gene from Streptococcus sp. Biochem J 289 (Pt 1), 179-84.
(63) Nagarajan, M., and Rao, V. S. (1979) Conformational studies of proteoglycans: theoretical studies on the conformation of heparin. Biopolymers 18, 1407-20.
(64) Lee, J. C, Lu, X. A., Kulkarni, S. S, Wen, Y. S., and Hung, S. C. (2004) Synthesis of heparin oligosaccharides. J Am Chem Soc 126, 476-7.
(65) Zhang, H., Muramatsu, T., Murase, A., Yuasa, S., Uchimura, K., and Kadomatsu, K. (2006) N-Acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury. Glycobiology 16, 702-10.
(66) Funderburgh, J. L. (2000) Keratan sulfate: structure, biosynthesis, and function. Glycobiology 10, 951-8.
(67) Ginsburg, V. (1978) Comparative biochemistry of nucleotide-linked sugars. Prog Clin Biol Res 23, 595-600.
(68) Plumbridge, J. A., Cochet, O., Souza, J. M., Altamirano, M. M., Calcagno, M. L., and Badet, B. (1993) Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12. J Bacteriol 175, 4951-6.
(69) Knop, J. K., and Hansen, R. G. (1970) Uridine diphosphate glucose pyrophosphorylase. IV. Crystallization and properties of the enzyme from human liver. J Biol Chem 245, 2499-504.
(70) Bulter, T., and Elling, L. (1999) Enzymatic synthesis of nucleotide sugars. Glycoconj J 16, 147-59.
(71) Cabib, E., Carminatti, H., and Woyskovsky, N. M. (1965) Phosphorolysis Of The Pyrophosphate Bond Of Sugar Nucleotides. Ii. Purification And Properties Of The Enzyme. J Biol Chem 240, 2114-21.
(72) Carminatti, H., and Cabib, E. (1965) Phosphorolysis Of The Pyrophosphate Bond Of Sugar Nucleotides. I. Characterization And Stoichiometry Of The Reaction. J Biol Chem 240,2110-3.
(73) Uehara, T., and Park, J. T. (2004) The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling. J Bacteriol 186, 7273-9.
(74) Lunin, V. V, Li, Y., Schrag, J. D., Iannuzzi, P., Cygler, M., and Matte, A. (2004) Crystal structures of Escherichia coli ATP-dependent glucokinase and its complex with glucose. J Bacteriol 186, 6915-27.
(75) Meyer, D., Schneider-Fresenius, C., Horlacher, R., Peist, R., and Boos, W. (1997) Molecular characterization of glucokinase from Escherichia coli K-12. J Bacteriol 179, 1298-306.
(76) Hofmann, M., Boles, E., and Zimmermann, F. K. (1994) Characterization of the essential yeast gene encoding N-acetylglucosamine-phosphate mutase. Eur J Biochem 221,741-7.
(77) Jolly, L., Ferrari, P., Blanot, D., Van Heijenoort, J., Fassy, F., and Mengin-Lecreulx, D. (1999) Reaction mechanism of phosphoglucosamine mutase from Escherichia coli. Eur J Biochem 262, 202-10.
(78) Mao, Z., Shin, H. D., and Chen, R. R. (2006) Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis. Biotechnol Prog 22,369-74.
(79) Abeijon, C., Robbins, P. W., and Hirschberg, C. B. (1996) Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis. Proc Natl Acad Sci U S A 93, 5963-8.
(80) Martinez-Duncker, I., Mollicone, R., Codogno, P., and Oriol, R. (2003) The nucleotide-sugar transporter family: a phylogenetic approach. Biochimie 85, 245-60.
(81) Berninsone, P., Miret, J. J., and Hirschberg, C. B. (1994) The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles. J Biol Chem 269, 207-11.
(82) Hirschberg, C. B., Robbins, P. W., and Abeijon, C. (1998) Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 67, 49-69.
(83) Kawakita, M., Ishida, N., Miura, N., Sun-Wada, G. H., and Yoshioka, S. (1998) Nucleotide sugar transporters: elucidation of their molecular identity and its implication for future studies. J Biochem 123, 777-85.
(84) Gerardy-Schahn, R., Oelmann, S., and Bakker, H. (2001) Nucleotide sugar transporters: biological and functional aspects. Biochimie 83, 775-82.
(85) Berninsone, P., and Hirschberg, C. B. (1998) Nucleotide sugars, nucleotide sulfate, and ATP transporters of the endoplasmic reticulum and Golgi apparatus. Ann N Y Acad Sci 842, 91-9.
(86) Yamamoto, K., Kawai, H., and Tochikura, T. (1981) Preparation of Uridine Diphosphate-N-Acetylgalactosamine from Uridine Diphosphate-N-Acetylglucosamine by Using Microbial Enzymes. Appl Environ Micwbiol 41, 392-395.
(87) Allard, S. T., Giraud, M. F., and Naismith, J. H. (2001) Epimerases: structure, function and mechanism. Cell Mol Life Sci 58, 1650-65.
(88) Dormann, P., and Benning, C. (1998) The role of UDP-glucose epimerase in carbohydrate metabolism of Arabidopsis. Plant J13, 641-52.
(89) Lopez, A. B., Sener, K., Trosien, J., Jarroll, E. L., and van Keulen, H. (2007) UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity. J Eukaryot Micwbiol 54, 154-60.
(90) Guo, H., Li, L., and Wang, P. G (2006) Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7. Biochemistry 45, 13760-8.
(91) Schulz, J. M., Watson, A. L., Sanders, R., Ross, K. L., Thoden, J. B., Holden, H.M.,and Fridovich-Keil,J.L.(2004) Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase.J Biol Chem 279,32796-803.
(92) Thoden JB,W.T.,Fridovich-Keil JL,Holden HM.(2000) Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase.Biochemistry 39,5691-5701.
(93) Guo,H.,Yi,W.,Li,L.,and Wang,P.G.(2007) Three UDP-hexose 4-epimerases with overlapping substrate specificity coexist in E.coli O86:B7.Biochem Biophys Res Commun 356,604-9.
(94) Chen,X.,Fang,J.,Zhang,J.,Liu,Z.,Shao,J.,Kowal,P.,Andreana,P.,and Wang,P.G.(2001) Sugar nucleotide regeneration beads(superbeads):a versatile tool for the practical synthesis of oligosaccharides.J Am Chem Soc 123,2081-2.
(95) Fang,J.,Chen,X.,Zhang,W.,Janczuk,A.,and Wang,P.G.(2000) Synthesis of alpha-Gal epitope derivatives with a galactosyltransferase-epimerase fusion enzyme.Carbohydr Res 329,873-8.
(96) Wu B,Z.Y.,Zheng R,Guo C,Wang PG.(2002) Bifunctional phosphomannose isomerase/GDP-D-mannose pyrophosphorylase is the point of control for GDP-D-mannose biosynthesis in Helicobacter pylori.FEBS Lett 519,87-92.
(97) Wu B,Z.Y.,Wang PG.(2001) Identification and characterization of GDP-d-mannose 4,6-dehydratase and GDP-1-fucose snthetase in a GDP-1-fucose biosynthetic gene cluster from Helicobacter pylori.Biochem Biophys Res Commun.285,364-371.
(98) Nakagawa,J.,Tamaki,S.,Tomioka,S.,and Matsuhashi,M.(1984) Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides.Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase.J Biol Chem 259,13937-46.
(99) Williamson,J.M.,Anderson,M.S.,and Raetz,C.R.(1991) Acyl-acyl carrier protein specificity ofUDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure. J Bacteriol 173, 3591-6.
(100) Kuhn, H. M., Meier-Dieter, U., and Mayer, H. (1988) ECA, the enterobacterial common antigen. FEMS Microbiol Rev 4,195-222.
(101) Chang, R., Moquist, P., and Finney, N. S. (2004) Chemical synthesis of UDP-4-O-methyl-GlcNAc, a potential chain terminator of chitin synthesis. Carbohydr Res 339,1531-6.
(102) Bulik, D. A., Olczak, M., Lucero, H. A., Osmond, B. C, Robbins, P. W., and Specht, C. A. (2003) Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress. Eukaryot Cell 2, 886-900.
(103) Herscovics, A., and Orlean, P. (1993) Glycoprotein biosynthesis in yeast. Faseb J 7, 540-50.
(104) Namboori, S. C., and Graham, D. E. (2008) Acetamido sugar biosynthesis in the Euryarchaea. J Bacteriol 190, 2987-96.
(105) Levy, P., and Picard, J. (1976) Glycosaminoglycan biosynthesis in arterial wall. Hexosaminyltransferase and glucuronyltransferase in cell membranes of aortic media-intima. Eur J Biochem 61, 613-9.
(106) Feng, F., Okuyama, K., Niikura, K., Ohta, T., Sadamoto, R., Monde, K., Noguchi, T., and Nishimura, S. (2004) Chemo-enzymatic synthesis of fluorinated 2-N-acetamidosugar nucleotides using UDP-GlcNAc pyrophosphorylase. Org Biomol Chem 2, 1617-23.
(107) Cecchelli, R., Cacan, R., and Verbert, A. (1985) Accumulation of UDP-GlcNAc into intracellular vesicles and occurrence of a carrier-mediated transport. Study with plasma-membrane-permeabilized mouse thymocytes. Eur J Biochem 153, 111-6.
(108) Sasai, K., Ikeda, Y., Fujii, T., Tsuda, T., and Taniguchi, N. (2002) UDP-GlcNAc concentration is an important factor in the biosynthesis of betal,6-branched oligosaccharides: regulation based on the kinetic properties of N-acetylglucosaminyltransferase V. Glycobiology 12, 119-27.
(109) Boehmelt, G, Wakeham, A., Elia, A., Sasaki, T., Plyte, S., Potter, J., Yang, Y., Tsang,E.,Ruland,J.,Iscove,N.N.,Dennis,J.W.,and Mak,T.W.(2000)Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32-deficient cells.Embo J 19,5092-104.
(110) Stokes,M.J.,Guther,M.L.,Turnock,D.C.,Prescott,A.R.,Martin,K.L.,Alphey,M.S.,and Ferguson,M.A.(2008) The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation.J Biol Chem 283,16147-61.
(111) Mio,T.,Yabe,T.,Arisawa,M.,and Yamada-Okabe,H.(1998) The eukaryotic UDP-N-acetylglucosamine pyrophosphorylases.Gene cloning,protein expression,and catalytic mechanism.J Biol Chem 273,14392-7.
(112) Milewski,S.,Gabriel,I.,and Olchowy,J.(2006) Enzymes of UDP-GlcNAc biosynthesis in yeast.Yeast 23,1-14.
(113) m.Huang,L.M.G.(2002)De novo synthesis of pyrimidine nucleotides;emerging interfaces with signal transduction pathways.Cell.Mol.Life Sci.60,321-336.
(114) Yamada-Okabe,T.,Sakamori,Y.,Mio,Y.,and Yamada-Okabe,H.(2001)Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.Eur J Biochem 268,2498-505.
(115) Shepherd,J.G.,Wang,L.,and Reeves,P.R.(2000) Comparison of O-antigen gene clusters of Escherichia coli(Shigella) sonnei and Plesiomonas shigelloides O17:sonnei gained its current plasmid-borne O-antigen genes from P.shigelloides in a recent event.Infect Immun 68,6056-61.
(116) Okuyama,K.,Hamamoto,T.,Ishige,K.,Takenouchi,K.,and Noguchi,T.(2000) An efficient method for production of uridine 5'-diphospho-N-acetylglucosamine.Biosci Biotechnol Biochem 64,386-92.
(117) Chang,R.,Vo,T.T.,and Finney,N.S.(2006) Synthesis of the C1-phosphonate analog of UDP-GlcNAc.Carbohydr Res 341,1998-2004.
(118) Schafer,A.,and Thiem,J.(2000) Synthesis of novel donor mimetics of UDP-Gal,UDP-GlcNAc,and UDP-GalNAc as potential transferase inhibitors.J Org Chem 65,24-9.
(119) Jurgen E.Heidlas,W.J.L.,Patrick Pale,George M.Whitesides.(1992)Gram-scales synthesis of Uridine 5'-Diphospho-N-acetylglucosamine:Comparison of Enzymatic and Chemical Routes.J.Org.Chem 57,146-151.
(120) Jungdon Bae,K.-H.K.,Dooil Kim,Yongseok Choi,Joong Su Kim,Sukhoon Hoh,Suk-In Hong,Dae-Sil Lee.(2005) A practical enzymatic synthesis of UDP-sugars and NDP Glucoses.Chembiochem 6,1963-1966.
(121) Leiting,B.,Pryor,K.D.,Eveland,S.S.,and Anderson,M.S.(1998) One-day enzymatic synthesis and purification of UDP-N-[1-14C]acetyl-glucosamine.Anal Biochem 256,185-91.
(122) Tochikura,Y.K.,H.,Gotan,T.(1971) studies on microbial metabolism of sugar nucleotides.Part Ⅵ.Agric.Biol.Chem.35,163-176.
(123) Jun Shao,J.Z.,Jozef Nahalka Peng George Wang.(2002) Biocatalytic synthesis of uridine 5'-diphosphate N-acetylglucosamine by multiple enzymes co-immobilized on agarose beads.Chem.Commun.,2586-2587.
(124) Koizumi,S.,Endo,T.,Tabata,K.,and Ozaki,A.(1998) Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria.Nat Biotechnol 16,847-50.
(125) Kazuhibo Tabata,S.K.,Tetsuo Endo,Akio Ozaki.(2000) Production of UDP-N-acetylglucosamine by coupling metabolically engineered bacteria.Biotechnology Letters 22,479-483.
(126) Gary C.Look,C.H.F.,Chi Huey Wong.(1993) Enzyme-catalyzed organic synthesis:practical routes to aza sugars and their analogs for use as glycprocessing inhibitors.Acc.Chem.Res.26,181-190.
(127) Sunthankar,P.,Pastuszak,I.,Rooke,A.,Elbein,A.D.,van de Rijn,I.,Canfield,W.M.,and Drake,R.R.(1998) Synthesis of 5-azido-UDP-N-acetylhexosamine photoaffinity analogs and radiolabeled UDP-N-acetylhexosamines.Anal Biochem 258,195-201.
(128) Pinney, K. G., Mejia, M. P., Villalobos, V. M., Rosenquist, B. E., Pettit, G R., Verdier-Pinard, P., and Hamel, E. (2000) Synthesis and biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin. Bioorg Med Chem 8, 2417-25.
(129) Jurgen E. Heidlas, W. J. L., Patrick Pale, George M. Whitesides. (1992) Gram-scale synthesis of uridine 5'-diphospho-N-acetylglucosamine: comparison of enzymic and chemical routes. J.Org.Chem 57,146-151.
(130) Togayachi, A., Akashima, T., Ookubo, R., Kudo, T., Nishihara, S., Iwasaki, H., Natsume, A., Mio, H., Inokuchi, J., Irimura, T., Sasaki, K., and Narimatsu, H. (2001) Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids. J Biol Chem 276, 22032-40.
(131) Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteornics 8, 3294-302.
(132) Kamst, E., Pilling, J., Raamsdonk, L. M., Lugtenberg, B. J., and Spaink, H. P. (1997) Rhizobium nodulation protein NodC is an important determinant of chitin oligosaccharide chain length in Nod factor biosynthesis. J Bacteriol 179, 2103-8.
(133) Chen, W. Y., Marcellin, E., Hung, J., and Nielsen, L. K. (2009) Hyaluronan molecular weight is controlled by UDP-N-acetylglucosamine concentration in Streptococcus zooepidemicus. J Biol Chem 284,18007-14.
(134) Lee, F. K., Stephens, D. S., Gibson, B. W., Engstrom, J. J., Zhou, D., and Apicella, M. A. (1995) Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB. Infect Immun 63, 2508-15.
(135) Hanover, J. A. (2001) Glycan-dependent signaling: O-linked N-acetylglucosamine. Faseb J 15,1865-76.
(136) Pattabiraman, T. N., and Bachhawat, B. K. (1961) Purification of uridine diphosphoacetylglucosamine pyrophosphorylase from sheep brain. Biochim Biophys Acta 50, 129-34.
(137) Szumilo, T., Zeng, Y, Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) Purification to homogeneity and properties of UDP-GlcNAc (GalNAc) pyrophosphorylase. J Biol Chem 271, 13147-54.
(138) Maruyama, D., Nishitani, Y., Nonaka, T., Kita, A., Fukami, T. A., Mio, T., Yamada-Okabe, H., Yamada-Okabe, T., and Miki, K. (2007) Crystal structure of uridine-diphospho-N-acetylglucosamine pyrophosphorylase from Candida albicans and catalytic reaction mechanism. J Biol Chem 282,17221-30.
(139) Bulik, D. A., van Ophem, P., Manning, J. M., Shen, Z., Newburg, D. S., and Jarroll, E. L. (2000) UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated. J Biol Chem 275, 14722-8.
(140) Mengin-Lecreulx, D., and van Heijenoort, J. (1993) Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli. J Bacteriol 175, 6150-7.
(141) Pompeo, F., Bourne, Y, van Heijenoort, J., Fassy, F., and Mengin-Lecreulx, D. (2001) Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth. J Biol Chem 276, 3833-9.
(142) Mengin-Lecreulx, D., and van Heijenoort, J. (1994) Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis. J Bacteriol 176, 5788-95.
(143) Mochalkin, I., Lightle, S., Zhu, Y, Ohren, J. F., Spessard, C., Chirgadze, N. Y., Banotai, C., Melnick, M., and McDowell, L. (2007) Characterization of substrate binding and catalysis in the potential antibacterial target N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). Protein Sci 16, 2657-66.
(144) Olsen, L. R., and Roderick, S. L. (2001) Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites. Biochemistry 40, 1913-21.
(145) Gehring, A. M., Lees, W. J., Mindiola, D. J., Walsh, C. T., and Brown, E. D. (1996) Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli. Biochemistry 35, 579-85.
(146) Parisi, G, Fornasari, M. S., and Echave, J. (2004) Dynactins p25 and p27 are predicted to adopt the LbetaH fold. FEBS Lett 562, 1-4.
(147) Parisi, G, and Echave, J. (2004) The structurally constrained protein evolution model accounts for sequence patterns of the LbetaH superfamily. BMC Evol Biol 4,41.
(148) Pompeo, F., van Heijenoort, J., and Mengin-Lecreulx, D. (1998) Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes. J Bacteriol 180, 4799-803.
(149) Olsen, L. R., Vetting, M. W., and Roderick, S. L. (2007) Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products. Protein Sci 16, 1230-5.
(150) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72,248-54.
(151) Zenker, A., Galanski, M., Bereuter, T. L., Keppler, B. K., and Lindner, W. (1999) Capillary electrophoretic study of cisplatin interaction with nucleoside monophosphates, di- and trinucleotides. J Chromatogr A 852, 337-46.
(152) Kostrewa, D., D'Arcy, A., Takacs, B., and Kamber, M. (2001) Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase,GlmU,in apo form at 2.33 A resolution and in complex with UDP-N-acetylglucosamine and Mg(2+) at 1.96 A resolution.J Mol Biol 305,279-89.
(153) Evans,C.E.,and Lovell,P.A.(2009) Click chemistry as a route to surface functionalization of polymer particles dispersed in aqueous media.Chem Commun(Camb),2305-7.
(154) Leavy,T.M.,and Bertozzi,C.R.(2007) A high-throughput assay for O-GlcNAc transferase detects primary sequence preferences in peptide substrates.Bioorg Med Chem Lett 17,3851-4.
(155) Hang,H.C.,Yu,C.,Kato,D.L.,and Bertozzi,C.R.(2003) Ametabolic labeling approach toward proteomic analysis ofmucin-type O-linked glycosylation.Proc Natl A cad Sci U S A 100,14846-51.
(156) Bond,M.R.,and Kohler,J.J.(2007) Chemical methods for glycoprotein discovery.Curr Opin Chem Biol 11,52-8.
(157) Vocadlo,D.J.,Hang,H.C.,Kim,E.J.,Hanover,J.A.,and Bertozzi,C.R.(2003) A chemical approach for identifying O-GlcNAc-modified proteins in cells.Proc Natl Acad Sci USA 100,9116-21.
(158) Guisheng Zhang,L.F.,Lizhi Zhu,Yanqiang Zhong,Peng George Wang,Duxin Sun.(2006) syntheses and biological activities of 3'-azido disaccharide analogues of daunorubicin against drug-resistant leukemia.J.Med.Chem.29,1792-1799.
(159) Saxon,E.,Luchansky,S.J.,Hang,H.C.,Yu,C.,Lee,S.C.,and Bertozzi,C.R.(2002) Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation.J Am Chem Soc 124,14893-902.
(160) Ruhela,D.,and Vishwakarma,R.A.(2003) Iterative synthesis of Leishmania phosphoglycans by solution,solid-phase,and polycondensation approaches without involving any glycosylation.J Org Chem 68,4446-56.
(161) Sven Fey,L.E.,Udo Kragl.(1997) The cofactor Mg2+--a key switch for effective continuous enzymatic production of GDP-mannose using recombinant GDP-mannose pyrophosphorylase.Carbohydrate Research 305, 475-481.
(162) Nishimoto,M.,and Kitaoka,M.(2007) Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum.Appl Environ Microbiol 73,6444-9.