氨甲酰基转移酶Asm21在安丝菌素合成中的双重催化功能研究
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摘要
美登素是强力抗癌药物,通过破坏微管组装发挥其细胞毒性。与抗体结合的靶向美登素分子的肿瘤选择性更强,半衰期更长,目前处于不同的临床实验期,发展势头良好。Actinosynnema pretiosum ssp. auranticum ATCC 31565产生的安丝菌素是微生物来源的美登素分子。在多个不同组分中,安丝菌素P-3是活性最强的主要组分,它的生物合成途径以3-氨基-5-羟基苯甲酸(AHBA)作为起始单元,随后在I型聚酮合酶的催化下加载7个延伸单元得到19元大环内酰胺proansamitocin,最后经过氯代、甲基化、酰基化、氨甲酰化、环氧化等一系列的后修饰反应得到安丝菌素。
近年来,从ATCC 31565的固体发酵产物中分离到了一系列安丝菌素糖苷AGP-1、AGP-2和AGP-3,它们在酰胺N的位置携带了β-D-葡萄糖基团。与此同时,Snipes等从Amycolatopsis sp. CP2808中也分离到了一系列携带氨甲酰化葡萄糖基的极性安丝菌素糖苷。最近,我们的合作者在ATCC 31565中同样发现了葡萄糖基C-4羟基发生氨甲酰化的安丝菌素糖苷(ACGP-3)。本研究组已经证实该糖基化反应是由N-糖基转移酶Asm25负责的,而这一系列氨甲酰化糖苷新产物的发现,促使我们去寻找负责葡萄糖氨甲酰化的基因和酶。
利用本研究组积累的多个安丝菌素生物合成基因缺失突变株,通过静息细胞转化实验,我们可以初步确定,糖苷葡萄糖基的氨甲酰化是由氨甲酰基转移酶Asm21来负责的。而在前期的研究中,通过序列同源性比对以及基因缺失实验,已经初步证明位于安丝菌素生物合成基因簇中的asm21负责C-7氨甲酰化和噁嗪环的形成。那么在安丝菌素的生物合成中,Asm21就具有双重催化的功能,不仅催化骨架的氨甲酰化,同时也催化葡萄糖基上的氨甲酰化,与其他次级代谢中的氨甲酰基转移酶有很大的不同,因此本研究对它进行了全面的研究。
通过基因失活和一系列的回补实验,不仅证明asm21确实参与了安丝菌素的合成,而且重新定义了asm21功能基因的长度。除了之前已经报道过的19-chloroproansamitocin之外,我们从asm21基因缺失突变株(BLQ16)的固体培养物中还分离得到了三个新的中间产物,并通过核磁共振分别确定了它们的结构。这三个新化合物分别为14-β-hydroxy-20-O-methyl-19-chloroisoproansamitocin、14- -hydroxy- 20-O-methyl-19-chloroisoproansamitocin和20-O-methyl-19- chloroproansamitocin,它们都缺失了C-7氨甲酰基。其中20-O-methyl- 19-chloroproansamitocin是主要产物,并被用作底物来检验异源表达的Asm21蛋白的酶学性质。以氨甲酰磷酸作为供体,在Mg2+和ATP存在的情况下,确定Asm21的最适温度、pH及金属离子分别为37℃、pH 8.5和10 mM Mg2+。Asm21对20-O-methyl-19-chloroproansamitocin和19-chloroproansamitocin的Km值分别为25.2±8.7μM和78.7±18.8μM,表明它对主要产物20-O-methyl-19-chloroproansamitocin的亲和力更强,以它为天然底物。
前期研究通过包涵体复性得到了Asm25的蛋白,而我们通过使用新的载体和表达条件,纯化得到了N-糖基转移酶Asm25的可溶活性蛋白,并通过酶促反应催化从N-去甲基安丝菌素P-3(PND-3)制备得到AGP-3,以此作为Asm21体外反应的底物。纯化的Asm21蛋白同样也可以催化AGP-3的氨甲酰化从而得到ACGP-3。Asm21对糖苷AGP-3的Km值为135.3±38.4μM,远远高于另外两个底物,表明Asm21更倾向与聚酮骨架结合。然而,Asm21对于糖苷的整体催化效率要高于聚酮骨架底物。最后,我们不仅在体外实现了Asm21的双重催化以及它与Asm25的串联催化,而且通过静息细胞体系将20-O-methyl-19-chloroproansamitocin同时转化为安丝菌素P-3及氨甲酰化的糖苷ACGP-3,在体内天然条件下也重现了这个过程。
本研究是第一次对具有高度底物灵活性的抗生素O-氨甲酰基转移酶进行的深入的生化研究,并在体内和体外同时实现了氨甲酰基转移酶的双重催化以及它与糖基转移酶的串联催化。值得注意的是,这个酶不仅催化大环内酰胺骨架上的C-7氨甲酰化,接受类似的安丝菌素中间产物为底物,同时也负责安丝菌素糖苷的C-4羟基的氨甲酰化。因此,安丝菌素的生物合成途径通过N-糖基转移酶Asm25和O-氨基甲酰转移酶Asm21的串联催化得到了延伸。由于其广泛的底物范围及氨甲酰基对于抗生素活性的重要性,Asm21可能被用来得到新的氨甲酰化抗生素衍生物,从而为新药物的开发提供工具。
安莎类抗生素及其他来源于AHBA(3-氨基-5-羟基苯甲酸)的抗生素在临床上具有非常重要的意义,它们分别具有很强的抗细菌、抗真菌或者抗肿瘤活性。在重要抗结核药物利福霉素的生物合成研究中,AHBA的合成途径得到了全面而深入的研究。其中AHBA合酶RifK催化最后一步从aDHS到AHBA的转化,而且这个蛋白与其他安莎类抗生素生物合成基因簇中的同源蛋白的相似性非常高。另一方面,以抗糖尿病药物阿卡波糖和抗真菌抗生素井冈霉素(有效霉素)为代表的C7N氨基环醇家族天然产物,它们都含有C7N氨基环醇的核心结构。这些化合物的生物合成途径共同起始于7-磷酸景天庚酮糖到2-表-5-表-有效醇酮的环化(2-epi-5-epi-valiolone),这个反应是由磷酸糖环化酶(井冈霉素中为ValA,阿卡波糖中为AcbC)催化的,之后2-表-5-表-有效醇酮经由不同的催化路径被转化为不同的终产物。不同基因簇中AHBA合酶和环化酶序列之间高度的保守性使得我们可以根据CODEHOP原则设计简并引物,然后从放线菌菌种库中筛选潜在的基因资源,为进一步得到新活性抗生素提供条件和基础。最终,从实验室小型菌种库中共获得7株AHBA合酶基因阳性菌株,3株环化酶基因阳性菌株,并通过克隆测序验证了扩增序列的正确性,比较了与已知基因的同源性。
从环化酶基因阳性菌株之一Streptomyces sp. CS中扩增得到的DNA片段所编码的氨基酸序列与ValA及AcbC的同源性都比较高,分别为58 %和52 %。随后,通过构建基因组文库和筛选,得到了13个包含环化酶探针片段的相互重叠的阳性fosmids。其中,选择16H9进行测序,通过Frameplot 3.0和BLASTp分别预测编码框和比对蛋白同源性,发现了20多个开放阅读框(ORF)。令人感兴趣的是,ORF16 - 20聚集成簇排列,分别与AcbC、AcbM、AcbL、AcbN及AcbO存在较高同源性,同时与这五个基因在阿卡波糖生物合成基因簇中的成簇分布是一样的。这五个基因催化阿卡波糖生物合成途径的前五步反应,也就是从7-磷酸景天庚酮糖合成7-磷酸-1-表-有效烯醇。7-磷酸-1-表-有效烯醇与先前报道的CS链霉菌中分离得到的C7多羟基环己烷衍生物CSS的结构非常接近。将基因簇中的CS-valA基因敲除后,CSS不再产生,表明这个基因簇可能负责它的生物合成。携带整个基因簇的18.3 kb基因片段在白色链霉菌中异源表达成功,进一步为该基因簇的功能提供了证据。从基因到天然产物的挖掘策略为新活性抗生素的挖掘提供了一条全新且可行的途径。
Maytansinoids are potent antitumor agents that exert their cytotoxicity by disrupting microtubule assembly. Antibody conjugates of maytansinoids exhibit increased tumour selectivity and longer circulation half-life and are currently in different stages of clinical development. Ansamitocins, produced by Actinosynnema pretiosum ssp. auranticum ATCC 31565, are maytansinoids of microbial origin. In the biosynthesis of ansamitocin P-3, 3-amino-5-hydroxybenzoic acid (AHBA) is used as a starter unit, and the incorporation of seven PKS extender units gives a 19-membered macrolactam, proansamitocin, which further undergoes a series of post-PKS modifications, including O- and N-methylation, chlorination, epoxidation, O-carbamoylation, and O-acylation.
Recently, ansamitocinosides P-1, P-2 and P-3 (AGP-1, AGP-2 and AGP-3) were isolated from A. pretiosum, which carry aβ-D-glucosyl moiety attached to the amide nitrogen in place of the N-methyl group of ansamitocins. Meanwhile, a series of polar ansacarbamitocins with a glucosyl moiety and three carbamoyl groups were isolated from Amycolatopsis sp. CP2808. Moreover, two new ansamitocin derivatives produced by mutational biosynthesis were assigned to be N-β-D-glucopyranosylated at the macrolactam amide. In addition, a novel ansamitocinoside with carbamoyl substitution at the C-4 hydroxyl group of the N-β-D-glucosyl moiety (ACGP-3) was identified from Actinosynnema pretiosum by our co-workers. Asm25 has been proved to be the dedicated N-glycosyltransferase for the sugar attachment. All these findings raised the question which genes and enzymes are responsible for the carbamoylation of the glucose hydroxyl group.
The construction of numerous mutants of A. pretiosum ATCC 31565 in the previous work allowed convenient identification ofthe relevant functional carbamoyltransferase gene(s). Through biotransformation, the carbamoyltransferase gene asm21 was suggested to be responsible for the carbamoylation of the glucosyl moiety. Moreover, based on sequence homology, gene asm21 in the biosynthetic gene cluster of ansamitocin was assigned the putative function of introducing the cyclic carbinolamide group. Therefore Asm21 was proved to have dual carbamoylation activityon both the polyketide backbone and the glucosyl moiety in the biosynthesis of ansamitocin. This is quite unique and interesting because the dual actions by one carbamoyltransferase have not been reported before.
Through gene inactivation and complementation, the involvement of asm21 in ansamitocin biosynthesis was confirmed. Besides, the functional length of gene asm21 was refined based on complementation results. In addition to the previously identified 19-chloroproansamitocin, three novel compounds (14-β-hydroxy-20-O-methyl-19-chloroiso proansamitocin,14- -hydroxy-20-O-methyl-19-chloroisoproansamitocin and 20-O-methyl-19-chloroproansamitocin) lacking the C-7 carbamoyl group were isolated from the culture of BLQ16 (asm21 mutant) on solid YMG medium and characterized by NMR. Among them, 20-O-methyl- 19-chloroproansamitocin was the major product and chosen to examine the enzymatic properties of Asm21 using carbamoyl phosphate as another substrate in the presence of Mg2+ and ATP.Asm21 was optimally active at 37oC and pH 8.5 - 9.0 and showed the highest activity when supplied with 5 - 10 mM Mg2+. It showed higher affinity towards 20-O-methyl- 19-chloroproansamitocin with a Km of 25.2±8.7μM compared to 19-chloroproansamitocinwith a Km of 78.7±18.8μM, indicating that 20-O-methyl-19- chloroproansamitocin is a preferred substrate for Asm21.
Asm25 was obtained as inclusion body in the previous report, and herein this study, soluble protein was expressed and purified with a different vector. Pure AGP-3 was therefore prepared through large scale incubation of PND-3 and UDP-glucose catalyzed by purified Asm25. Purified Asm21 also catalyzed the conversion of AGP-3 into ACGP-3, verifying that it does have dual carbamoylation activity on both the polyketide backbone and the sugar moiety. Moreover, Asm21 has a Km of 135.3±38.4μM for AGP-3, much higher than those for 20-O-methyl- 19-chloroproansamitocin or 19-chloroproansamitocin, which indicates a favored binding toward the polyketide backbone rather than the glucosyl moiety in the reaction site of Asm21. However, the overall catalytic efficiency (kcat /Km) for AGP-3 is higher than the other two substrates. Hence, the catalytic constants indicate that Asm21 displays a preference for carbamoylation of the glucosyl moiety over the polyketide backbone. Furthermore, the dual carbamoylations and N-glycosylation were precisely demonstrated in vivo.
This work represents the first biochemical characterization of an O-carbamoyltransferase with very high substrate flexibility during ansamitocin biosynthesis. Remarkably, the enzyme catalyzes not only the C-7 carbamoylation of the macrolactam backbone, accepting a variety of ansamitocin structures as substrates, but also the carbamoylation of the C-4 hydroxyl group of the N-glucosyl moiety in ansamitocinoside P-3. Thus, the ansamitocin biosynthetic pathway could be extended through the tandem catalysis of the N-glycosyltransferase Asm25 and the O-carbamoyltransferase Asm21 by cultivating the strain on solid medium. Due to its broad substrate range, Asm21 can be used to generate O-carbamoylated derivatives of many ansamycins as potential drug candidates.
The clinically important family of AHBA (3-amino-5-hydroxybenzoic acid) containing natural products, including ansamycins or other antibiotics, possesses potent antibacterial, antifungal or antitumor activities. The biosynthetic pathway of AHBA has been disclosed in the biosynthesis of rifamycin. AHBA synthase RifK catalyzes the last step converting aminoDHS into AHBA, and this protein shares high identity with its homologs in other ansamycin producers. On the other hand, the C7N-aminocyclitol family of natural products, exemplified by anti-diabetic acarbose and anti-fungal validamycin A, contain a C7N aminocyclitol in their core structure. The biosynthesis of these compounds is universally initiated with the cyclization of sedoheptulose 7-phosphate, catalyzed by a sugar phosphate cyclase (ValA for validamycin A and AcbC for acarbose), to give 2-epi-5-epi-valiolone, which then undergoes different routes to yield various final products. The high similarities shared by AHBA synthases and cyclases isolated from different gene clusters enabled us to design degenerate primers using CODEHOP to screen a pool of Actinomycete strains. As a result, seven AHBA positive and three cyclase positive strains were obtained from the laboratory library. The amplified DNA fragments were cloned and sequenced to confirm the correctness and homology with the probe。
The deduced animo acid sequence of onecyclase positive strain, Streptomyces sp. CS, showed high similarity to ValA and AcbC. Subsequently a fosmid genomic library was constructed and 13 fosmids were fished out with the cyclase primers. One of them (16H9) was fully sequenced, and more than 20 ORFs were identified using Frameplot 3.0 and BLASTp. Interestingly, ORF16 - 20, which showed high homology to AcbC, AcbM, AcbL, AcbN, and AcbO respectively, are clustered as in acarbose biosynthetic gene cluster. These five enzymes probably catalyze the first five reactions of the biosynthesis of acarbose to generate 1-epi- valienol 7-phosphate, whose chemical structure is quite similar to CSS, a polyhydroxyl derivative of C7 cyclohexane previously isolated from Streptomyces sp.CS. The production of CSS was abolished by inactivation of CS-valA, indicating that this gene cluster is responsible for its biosynthesis. This was further confirmed by a successful heterologous expression of the CSS gene cluster in S. albus J1074. This gene to compound mining strategy provides a new and pratical way for the discovery of new antibiotics.
近年来,从ATCC 31565的固体发酵产物中分离到了一系列安丝菌素糖苷AGP-1、AGP-2和AGP-3,它们在酰胺N的位置携带了β-D-葡萄糖基团。与此同时,Snipes等从Amycolatopsis sp. CP2808中也分离到了一系列携带氨甲酰化葡萄糖基的极性安丝菌素糖苷。最近,我们的合作者在ATCC 31565中同样发现了葡萄糖基C-4羟基发生氨甲酰化的安丝菌素糖苷(ACGP-3)。本研究组已经证实该糖基化反应是由N-糖基转移酶Asm25负责的,而这一系列氨甲酰化糖苷新产物的发现,促使我们去寻找负责葡萄糖氨甲酰化的基因和酶。
利用本研究组积累的多个安丝菌素生物合成基因缺失突变株,通过静息细胞转化实验,我们可以初步确定,糖苷葡萄糖基的氨甲酰化是由氨甲酰基转移酶Asm21来负责的。而在前期的研究中,通过序列同源性比对以及基因缺失实验,已经初步证明位于安丝菌素生物合成基因簇中的asm21负责C-7氨甲酰化和噁嗪环的形成。那么在安丝菌素的生物合成中,Asm21就具有双重催化的功能,不仅催化骨架的氨甲酰化,同时也催化葡萄糖基上的氨甲酰化,与其他次级代谢中的氨甲酰基转移酶有很大的不同,因此本研究对它进行了全面的研究。
通过基因失活和一系列的回补实验,不仅证明asm21确实参与了安丝菌素的合成,而且重新定义了asm21功能基因的长度。除了之前已经报道过的19-chloroproansamitocin之外,我们从asm21基因缺失突变株(BLQ16)的固体培养物中还分离得到了三个新的中间产物,并通过核磁共振分别确定了它们的结构。这三个新化合物分别为14-β-hydroxy-20-O-methyl-19-chloroisoproansamitocin、14- -hydroxy- 20-O-methyl-19-chloroisoproansamitocin和20-O-methyl-19- chloroproansamitocin,它们都缺失了C-7氨甲酰基。其中20-O-methyl- 19-chloroproansamitocin是主要产物,并被用作底物来检验异源表达的Asm21蛋白的酶学性质。以氨甲酰磷酸作为供体,在Mg2+和ATP存在的情况下,确定Asm21的最适温度、pH及金属离子分别为37℃、pH 8.5和10 mM Mg2+。Asm21对20-O-methyl-19-chloroproansamitocin和19-chloroproansamitocin的Km值分别为25.2±8.7μM和78.7±18.8μM,表明它对主要产物20-O-methyl-19-chloroproansamitocin的亲和力更强,以它为天然底物。
前期研究通过包涵体复性得到了Asm25的蛋白,而我们通过使用新的载体和表达条件,纯化得到了N-糖基转移酶Asm25的可溶活性蛋白,并通过酶促反应催化从N-去甲基安丝菌素P-3(PND-3)制备得到AGP-3,以此作为Asm21体外反应的底物。纯化的Asm21蛋白同样也可以催化AGP-3的氨甲酰化从而得到ACGP-3。Asm21对糖苷AGP-3的Km值为135.3±38.4μM,远远高于另外两个底物,表明Asm21更倾向与聚酮骨架结合。然而,Asm21对于糖苷的整体催化效率要高于聚酮骨架底物。最后,我们不仅在体外实现了Asm21的双重催化以及它与Asm25的串联催化,而且通过静息细胞体系将20-O-methyl-19-chloroproansamitocin同时转化为安丝菌素P-3及氨甲酰化的糖苷ACGP-3,在体内天然条件下也重现了这个过程。
本研究是第一次对具有高度底物灵活性的抗生素O-氨甲酰基转移酶进行的深入的生化研究,并在体内和体外同时实现了氨甲酰基转移酶的双重催化以及它与糖基转移酶的串联催化。值得注意的是,这个酶不仅催化大环内酰胺骨架上的C-7氨甲酰化,接受类似的安丝菌素中间产物为底物,同时也负责安丝菌素糖苷的C-4羟基的氨甲酰化。因此,安丝菌素的生物合成途径通过N-糖基转移酶Asm25和O-氨基甲酰转移酶Asm21的串联催化得到了延伸。由于其广泛的底物范围及氨甲酰基对于抗生素活性的重要性,Asm21可能被用来得到新的氨甲酰化抗生素衍生物,从而为新药物的开发提供工具。
安莎类抗生素及其他来源于AHBA(3-氨基-5-羟基苯甲酸)的抗生素在临床上具有非常重要的意义,它们分别具有很强的抗细菌、抗真菌或者抗肿瘤活性。在重要抗结核药物利福霉素的生物合成研究中,AHBA的合成途径得到了全面而深入的研究。其中AHBA合酶RifK催化最后一步从aDHS到AHBA的转化,而且这个蛋白与其他安莎类抗生素生物合成基因簇中的同源蛋白的相似性非常高。另一方面,以抗糖尿病药物阿卡波糖和抗真菌抗生素井冈霉素(有效霉素)为代表的C7N氨基环醇家族天然产物,它们都含有C7N氨基环醇的核心结构。这些化合物的生物合成途径共同起始于7-磷酸景天庚酮糖到2-表-5-表-有效醇酮的环化(2-epi-5-epi-valiolone),这个反应是由磷酸糖环化酶(井冈霉素中为ValA,阿卡波糖中为AcbC)催化的,之后2-表-5-表-有效醇酮经由不同的催化路径被转化为不同的终产物。不同基因簇中AHBA合酶和环化酶序列之间高度的保守性使得我们可以根据CODEHOP原则设计简并引物,然后从放线菌菌种库中筛选潜在的基因资源,为进一步得到新活性抗生素提供条件和基础。最终,从实验室小型菌种库中共获得7株AHBA合酶基因阳性菌株,3株环化酶基因阳性菌株,并通过克隆测序验证了扩增序列的正确性,比较了与已知基因的同源性。
从环化酶基因阳性菌株之一Streptomyces sp. CS中扩增得到的DNA片段所编码的氨基酸序列与ValA及AcbC的同源性都比较高,分别为58 %和52 %。随后,通过构建基因组文库和筛选,得到了13个包含环化酶探针片段的相互重叠的阳性fosmids。其中,选择16H9进行测序,通过Frameplot 3.0和BLASTp分别预测编码框和比对蛋白同源性,发现了20多个开放阅读框(ORF)。令人感兴趣的是,ORF16 - 20聚集成簇排列,分别与AcbC、AcbM、AcbL、AcbN及AcbO存在较高同源性,同时与这五个基因在阿卡波糖生物合成基因簇中的成簇分布是一样的。这五个基因催化阿卡波糖生物合成途径的前五步反应,也就是从7-磷酸景天庚酮糖合成7-磷酸-1-表-有效烯醇。7-磷酸-1-表-有效烯醇与先前报道的CS链霉菌中分离得到的C7多羟基环己烷衍生物CSS的结构非常接近。将基因簇中的CS-valA基因敲除后,CSS不再产生,表明这个基因簇可能负责它的生物合成。携带整个基因簇的18.3 kb基因片段在白色链霉菌中异源表达成功,进一步为该基因簇的功能提供了证据。从基因到天然产物的挖掘策略为新活性抗生素的挖掘提供了一条全新且可行的途径。
Maytansinoids are potent antitumor agents that exert their cytotoxicity by disrupting microtubule assembly. Antibody conjugates of maytansinoids exhibit increased tumour selectivity and longer circulation half-life and are currently in different stages of clinical development. Ansamitocins, produced by Actinosynnema pretiosum ssp. auranticum ATCC 31565, are maytansinoids of microbial origin. In the biosynthesis of ansamitocin P-3, 3-amino-5-hydroxybenzoic acid (AHBA) is used as a starter unit, and the incorporation of seven PKS extender units gives a 19-membered macrolactam, proansamitocin, which further undergoes a series of post-PKS modifications, including O- and N-methylation, chlorination, epoxidation, O-carbamoylation, and O-acylation.
Recently, ansamitocinosides P-1, P-2 and P-3 (AGP-1, AGP-2 and AGP-3) were isolated from A. pretiosum, which carry aβ-D-glucosyl moiety attached to the amide nitrogen in place of the N-methyl group of ansamitocins. Meanwhile, a series of polar ansacarbamitocins with a glucosyl moiety and three carbamoyl groups were isolated from Amycolatopsis sp. CP2808. Moreover, two new ansamitocin derivatives produced by mutational biosynthesis were assigned to be N-β-D-glucopyranosylated at the macrolactam amide. In addition, a novel ansamitocinoside with carbamoyl substitution at the C-4 hydroxyl group of the N-β-D-glucosyl moiety (ACGP-3) was identified from Actinosynnema pretiosum by our co-workers. Asm25 has been proved to be the dedicated N-glycosyltransferase for the sugar attachment. All these findings raised the question which genes and enzymes are responsible for the carbamoylation of the glucose hydroxyl group.
The construction of numerous mutants of A. pretiosum ATCC 31565 in the previous work allowed convenient identification ofthe relevant functional carbamoyltransferase gene(s). Through biotransformation, the carbamoyltransferase gene asm21 was suggested to be responsible for the carbamoylation of the glucosyl moiety. Moreover, based on sequence homology, gene asm21 in the biosynthetic gene cluster of ansamitocin was assigned the putative function of introducing the cyclic carbinolamide group. Therefore Asm21 was proved to have dual carbamoylation activityon both the polyketide backbone and the glucosyl moiety in the biosynthesis of ansamitocin. This is quite unique and interesting because the dual actions by one carbamoyltransferase have not been reported before.
Through gene inactivation and complementation, the involvement of asm21 in ansamitocin biosynthesis was confirmed. Besides, the functional length of gene asm21 was refined based on complementation results. In addition to the previously identified 19-chloroproansamitocin, three novel compounds (14-β-hydroxy-20-O-methyl-19-chloroiso proansamitocin,14- -hydroxy-20-O-methyl-19-chloroisoproansamitocin and 20-O-methyl-19-chloroproansamitocin) lacking the C-7 carbamoyl group were isolated from the culture of BLQ16 (asm21 mutant) on solid YMG medium and characterized by NMR. Among them, 20-O-methyl- 19-chloroproansamitocin was the major product and chosen to examine the enzymatic properties of Asm21 using carbamoyl phosphate as another substrate in the presence of Mg2+ and ATP.Asm21 was optimally active at 37oC and pH 8.5 - 9.0 and showed the highest activity when supplied with 5 - 10 mM Mg2+. It showed higher affinity towards 20-O-methyl- 19-chloroproansamitocin with a Km of 25.2±8.7μM compared to 19-chloroproansamitocinwith a Km of 78.7±18.8μM, indicating that 20-O-methyl-19- chloroproansamitocin is a preferred substrate for Asm21.
Asm25 was obtained as inclusion body in the previous report, and herein this study, soluble protein was expressed and purified with a different vector. Pure AGP-3 was therefore prepared through large scale incubation of PND-3 and UDP-glucose catalyzed by purified Asm25. Purified Asm21 also catalyzed the conversion of AGP-3 into ACGP-3, verifying that it does have dual carbamoylation activity on both the polyketide backbone and the sugar moiety. Moreover, Asm21 has a Km of 135.3±38.4μM for AGP-3, much higher than those for 20-O-methyl- 19-chloroproansamitocin or 19-chloroproansamitocin, which indicates a favored binding toward the polyketide backbone rather than the glucosyl moiety in the reaction site of Asm21. However, the overall catalytic efficiency (kcat /Km) for AGP-3 is higher than the other two substrates. Hence, the catalytic constants indicate that Asm21 displays a preference for carbamoylation of the glucosyl moiety over the polyketide backbone. Furthermore, the dual carbamoylations and N-glycosylation were precisely demonstrated in vivo.
This work represents the first biochemical characterization of an O-carbamoyltransferase with very high substrate flexibility during ansamitocin biosynthesis. Remarkably, the enzyme catalyzes not only the C-7 carbamoylation of the macrolactam backbone, accepting a variety of ansamitocin structures as substrates, but also the carbamoylation of the C-4 hydroxyl group of the N-glucosyl moiety in ansamitocinoside P-3. Thus, the ansamitocin biosynthetic pathway could be extended through the tandem catalysis of the N-glycosyltransferase Asm25 and the O-carbamoyltransferase Asm21 by cultivating the strain on solid medium. Due to its broad substrate range, Asm21 can be used to generate O-carbamoylated derivatives of many ansamycins as potential drug candidates.
The clinically important family of AHBA (3-amino-5-hydroxybenzoic acid) containing natural products, including ansamycins or other antibiotics, possesses potent antibacterial, antifungal or antitumor activities. The biosynthetic pathway of AHBA has been disclosed in the biosynthesis of rifamycin. AHBA synthase RifK catalyzes the last step converting aminoDHS into AHBA, and this protein shares high identity with its homologs in other ansamycin producers. On the other hand, the C7N-aminocyclitol family of natural products, exemplified by anti-diabetic acarbose and anti-fungal validamycin A, contain a C7N aminocyclitol in their core structure. The biosynthesis of these compounds is universally initiated with the cyclization of sedoheptulose 7-phosphate, catalyzed by a sugar phosphate cyclase (ValA for validamycin A and AcbC for acarbose), to give 2-epi-5-epi-valiolone, which then undergoes different routes to yield various final products. The high similarities shared by AHBA synthases and cyclases isolated from different gene clusters enabled us to design degenerate primers using CODEHOP to screen a pool of Actinomycete strains. As a result, seven AHBA positive and three cyclase positive strains were obtained from the laboratory library. The amplified DNA fragments were cloned and sequenced to confirm the correctness and homology with the probe。
The deduced animo acid sequence of onecyclase positive strain, Streptomyces sp. CS, showed high similarity to ValA and AcbC. Subsequently a fosmid genomic library was constructed and 13 fosmids were fished out with the cyclase primers. One of them (16H9) was fully sequenced, and more than 20 ORFs were identified using Frameplot 3.0 and BLASTp. Interestingly, ORF16 - 20, which showed high homology to AcbC, AcbM, AcbL, AcbN, and AcbO respectively, are clustered as in acarbose biosynthetic gene cluster. These five enzymes probably catalyze the first five reactions of the biosynthesis of acarbose to generate 1-epi- valienol 7-phosphate, whose chemical structure is quite similar to CSS, a polyhydroxyl derivative of C7 cyclohexane previously isolated from Streptomyces sp.CS. The production of CSS was abolished by inactivation of CS-valA, indicating that this gene cluster is responsible for its biosynthesis. This was further confirmed by a successful heterologous expression of the CSS gene cluster in S. albus J1074. This gene to compound mining strategy provides a new and pratical way for the discovery of new antibiotics.
引文
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