蓝藻硫酯酶催化多肽环化适用性分析
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摘要
硫酯酶(thioesterase,TE)具有区域定向性(regiospecific)、化学定向性(chemospecific)及立体定向性(stereospecific)的特点。这些特性决定了TE作为生物催化剂(biocatalysis)在工业生产中具有较高的应用价值和广阔的应用前景。Mcy C TE(microcystin thioesterase,Mcy C TE)来自铜绿微囊藻(microcystis aeruginosa)NRPS/PKS生物合成基因簇。我们利用正交试验提高Mcy C TE表达量,得到稳定的诱导表达条件,并结合成熟的线性多肽化学合成法对其底物适用性做了进一步研究。得到的最佳诱导表达条件为:诱导时机2 h,诱导剂异丙基-β-D-硫代半乳糖苷(isopropyl-β-Dthiogalactopyranoside,IPTG)浓度0.75 mmol/L,诱导时间6 h,诱导转速210 r/min,诱导温度20℃,使TE的表达量由8.75 mg/L提高至22.15 mg/L,时间缩短了6.5 h。TE表达量的大幅度提升和表达时间的缩短为将来酶的结构及催化机制研究奠定了基础。TE底物适用性研究结果发现:Mcy C TE并不遵循"4 n+2原则";底物中转角过多不仅不利于环肽的形成,更可能形成卷曲影响环化;无D型氨基酸亦可通过加入其它位阻较小较灵活的Gly或者自带天然转角Pro的可弱化肽链的刚性,促进催化反应;含苯环的Phe的引入在一定程度上阻碍了环化;底物无肽链氨基酸数目奇偶性的选择;延长多肽链长度也可环化,Mcy C TE的底物容忍度较大,使天然多肽药物筛选范围增大,也为增强天然多肽药物药效增加了改良方案,为进一步研究Mcy C TE的催化功能提供了实验基础。
Thioesterase( TE) shows impressive stereospecificity,regioselectivity and chemoselectivity during the cyclization of peptide. The biocatalysis property of TE provides high value and extensive application in industrial production. We used the orthogonal experiment to further increase the expression of Mcy C TE and analyzed its substrate suitability by the synthesis of mature linear peptide chemistry.Finally the best induction condition was detected as follows: after incubation for 2 hours,0. 75 mmol / L IPTG was added and the culture was shaked at 210 r / min and 20 ℃ for 6 hours. The protein levels of TEwere improved from 8. 75 mg / L to 22. 15 mg / L and the induction time reduced 6. 5 hours. The improvement of TE expression quantity and induction time would provide clues for the future research of the enzyme structure and catalytic mechanism. The results showed that the Mcy C TE does not follow the principle of " 4 n + 2" or parity selection of the number of peptide chains of amino acids. Over-rotation is more likely to form curling which affects cyclization. Amino acids with lower steric hindrance and flexibility,such as Gly,or amino acids providing natural angle,such as Pro,could reduce the rigidity of the peptide chain to promote induction. The introduction of the Phe containing benzene ring inhibited cyclization. The study proved that extended the length of polypeptide chains could also induce cyclization and the Mcy C TE substrate tolerance was broadened. These results expanded the screening scope of natural peptide drugs,which provided the experimental basis for the further research of Mcy C TE catalytic function which may improve the efficacy of natural peptide drug.
Thioesterase( TE) shows impressive stereospecificity,regioselectivity and chemoselectivity during the cyclization of peptide. The biocatalysis property of TE provides high value and extensive application in industrial production. We used the orthogonal experiment to further increase the expression of Mcy C TE and analyzed its substrate suitability by the synthesis of mature linear peptide chemistry.Finally the best induction condition was detected as follows: after incubation for 2 hours,0. 75 mmol / L IPTG was added and the culture was shaked at 210 r / min and 20 ℃ for 6 hours. The protein levels of TEwere improved from 8. 75 mg / L to 22. 15 mg / L and the induction time reduced 6. 5 hours. The improvement of TE expression quantity and induction time would provide clues for the future research of the enzyme structure and catalytic mechanism. The results showed that the Mcy C TE does not follow the principle of " 4 n + 2" or parity selection of the number of peptide chains of amino acids. Over-rotation is more likely to form curling which affects cyclization. Amino acids with lower steric hindrance and flexibility,such as Gly,or amino acids providing natural angle,such as Pro,could reduce the rigidity of the peptide chain to promote induction. The introduction of the Phe containing benzene ring inhibited cyclization. The study proved that extended the length of polypeptide chains could also induce cyclization and the Mcy C TE substrate tolerance was broadened. These results expanded the screening scope of natural peptide drugs,which provided the experimental basis for the further research of Mcy C TE catalytic function which may improve the efficacy of natural peptide drug.
引文
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[14]Kohli RM,Trauger JW,Schwarzer D,et al.Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases[J].Biochemistry,2001,40(24):7099-7108
[15]Kohli R M,Walsh C T,Burkart M D.Biomimetic synthesis and optimization of cyclic peptide antibiotics[J].Nature,2002,418(6898):658-661
[16]Bruner SD,Weber T,Kohli RM,et al.Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain Srf TE[J].Structure,2002,10(3):301-310
[17]Kohli R M,Walsh C T.Enzymology of acyl chain macrocyclization in natural product biosynthesis[J].Chem Commun(Camb),2003,2003(3):297-307
[18]Samel S A,Wagner B,Marahiel M A,et al.The thioesterase domain of the fengycin biosynthesis cluster:a structural base for the macrocyclization of a non-ribosomal lipopeptide[J].J Mol Biol,2006,359(4):876-889
[19]Gehret JJ,Gu L,Gerwick WH,et al.Terminal alkene formation by the thioesterase of curacin A biosynthesis:structure of a decarboxylating thioesterase[J].J Biol Chem,2011,286(16):14445-14454
[20]Tseng CC,Bruner SD,Kohli RM,et al.Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase[J].Biochemistry,2002,41(45):13350-13359
[21]Trauger JW,Kohli RM,Walsh CT.Cyclization of backbonesubstituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase[J].Biochemistry,2001,40(24):7092-7098
[22]Gibbs AC,Kondejewski LH,Gronwald W,et al.Unusual betasheet periodicity in small cyclic peptides[J].Nat Struct Biol,1998,5(4):284-288
[2]Horsman ME,Hari TP,Boddy CN.Polyketide synthase and nonribosomal peptide synthetase thioesterase selectivity:logic gate or a victim of fate?[J].Nat Prod Rep,2016,33(2):183-202
[3]Gehring AM,Demoll E,Fetherston JD,et al.Iron acquisition in plague:modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis[J].Chem Biol,1998,5(10):573-586
[4]Keating TA,Ehmann D E,Kohli R M,et al.Chain termination steps in nonribosomal peptide synthetase assembly lines:directed acyl-S-enzyme breakdown in antibiotic and siderophore biosynthesis[J].Chembiochem,2001,2(2):99-107
[5]Staunton J,Wilkinson B.Biosynthesis of erythromycin and rapamycin[J].Chem Rev,1997,97(7):2611-2630
[6]Konz D,Klens A,Schorqendorfer K,et al.The bacitracin biosynthesis operon of Bacillus licheni formis ATCC 10716:molecular characterization of three multimodular peptide synthetases[J].Chem Biol,1997,4(12):927-937
[7]方剑,何魁芳,季赛,等.微囊藻毒素衍生物的化学酶法合成及活性评价[J].中国生物化学与分子生物学报(Fang J,He KF,Ji S,et al.The chemoenzymatic sythesis and bioactivity study of microcystin analogue[J].Chin J Biochem Mol Biol),2014,30(7):706-713
[8]Ruoslahti E.RGD and other recognition sequences for integrins[J].Annu Rev Cell Dev Biol,1996,12:697-715
[9]Humphries J D,Askari JA,Zhang XP,et al.Molecular basis of ligand recognition by integrin alpha5beta 1.II.Specificity of arggly-Asp binding is determined by Trp157 of the alpha subunit[J].J Biol Chem,2000,275(27):20337-20345
[10]Sainis I,Fokas D,Vareli K,et al.Cyanobacterial cyclopeptides as lead compounds to novel targeted cancer drugs[J].Mar Grugs,2010,8(3):629-657
[11]Trauger JW,Kohli RM,Mootz HD,et al.Peptides cyclization catalysed by the thioesterase domain from tyrocidine synthetase[J].Nature,2000,407(6801):215-218
[12]Giessen TW,Marahiel MA.Ribosome-independent biosynthesis of biologically active peptides:Application of synthetic biology to generate structural diversity[J].FEBS Lett,2012,586(15):2065-2075
[13]Grünewald J,Sieber S A,Mahlert C,et al.Synthesis and derivatization of daptomycin:a chemoenzymatic route to acidic lipopeptide antibiotics[J].J Am Chem Soc,2004,126(51):17025-17031
[14]Kohli RM,Trauger JW,Schwarzer D,et al.Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases[J].Biochemistry,2001,40(24):7099-7108
[15]Kohli R M,Walsh C T,Burkart M D.Biomimetic synthesis and optimization of cyclic peptide antibiotics[J].Nature,2002,418(6898):658-661
[16]Bruner SD,Weber T,Kohli RM,et al.Structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain Srf TE[J].Structure,2002,10(3):301-310
[17]Kohli R M,Walsh C T.Enzymology of acyl chain macrocyclization in natural product biosynthesis[J].Chem Commun(Camb),2003,2003(3):297-307
[18]Samel S A,Wagner B,Marahiel M A,et al.The thioesterase domain of the fengycin biosynthesis cluster:a structural base for the macrocyclization of a non-ribosomal lipopeptide[J].J Mol Biol,2006,359(4):876-889
[19]Gehret JJ,Gu L,Gerwick WH,et al.Terminal alkene formation by the thioesterase of curacin A biosynthesis:structure of a decarboxylating thioesterase[J].J Biol Chem,2011,286(16):14445-14454
[20]Tseng CC,Bruner SD,Kohli RM,et al.Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase[J].Biochemistry,2002,41(45):13350-13359
[21]Trauger JW,Kohli RM,Walsh CT.Cyclization of backbonesubstituted peptides catalyzed by the thioesterase domain from the tyrocidine nonribosomal peptide synthetase[J].Biochemistry,2001,40(24):7092-7098
[22]Gibbs AC,Kondejewski LH,Gronwald W,et al.Unusual betasheet periodicity in small cyclic peptides[J].Nat Struct Biol,1998,5(4):284-288