前导肽切割位点对Ⅱ类羊毛硫细菌素切割酶活性的影响
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摘要
【背景】Ⅱ类羊毛硫细菌素大多是由革兰氏阳性菌的核糖体合成并经过翻译后修饰产生的小肽,其生物合成的最后一步是由转运蛋白LanTN端的肽酶结构域对前导肽进行切割,释放出有活性的羊毛硫细菌素,但目前关于该类羊毛硫细菌素前导肽的切割机制尚不清楚。【目的】考察前导肽切割位点对不同链球菌来源的肽酶结构域BovT150和SboT150酶切活性的影响。【方法】运用不依赖连接酶的定点突变技术构建前导肽切割位点突变的前体蛋白表达载体,在大肠杆菌(Escherichia coli)中分别表达纯化野生型前体(Bov Am和Sbo Am)、突变型前体及对应的切割酶(Bov T150和Sbo T150),构建体外酶切体系,利用HPLC、抑菌活性分析和MALDI-TOF MS检测前导肽的切除情况。【结果】BovT150不仅能够切割Bov Am的GG和GA位点,也能切割Sbo Am的GG和GA位点,并且对切割位点为Gly的前体切割活性较高;Sbo T150仅能切割Sbo Am的GG和GA位点,而对切割位点为Ala的活性较高。【结论】II类羊毛硫细菌素前导肽切割位点氨基酸残基的改变不同程度地影响切割酶的切割效率。
[Background] Class Ⅱ lantibiotics are ribosomally synthesized and post-translationally modified peptides, mainly produced by Gram-positive bacteria. In the last step of its biosynthesis, the N-terminal peptidase domain of transporter protein LanT cleaves the leader peptide to produce active lantibiotics. However, the removal mechanism of leader peptide in this class lantibiotics is still not clear. [Objective] To investigate the effects of cleavage sites on the activity of peptidases domain BovT150 and SboT150 from different streptococci. [Methods] Expression vectors for the precursor peptides with mutated cleavage sites were constructed by site-directed ligase-independent mutagenesis and then the wild-type precursors(BovAm and SboAm), their mutant precursors, as well as the corresponding peptidases(BovT150 and SboT150) were expressed and purified in E. coli. The precursors were separately incubated with each peptidase in vitro and the removal efficiencies of the leader peptides were assessed by HPLC, antimicrobial activity assay and MALDI-TOF MS. [Results] Both cleavage sites GG and GA of BovAm and SboAm allowed BovT150 to retain peptidase activity, and Gly was more suitable to be processed by BovT150. Only cleavage sites GG and GA of SboAm were accessible to SboT150, which cleaved Ala more efficiently. [Conclusion] The change of amino acid residues at the cleavage sites of leader peptides affected the efficiencies of class Ⅱ lantibiotic peptidases in varying degrees.
[Background] Class Ⅱ lantibiotics are ribosomally synthesized and post-translationally modified peptides, mainly produced by Gram-positive bacteria. In the last step of its biosynthesis, the N-terminal peptidase domain of transporter protein LanT cleaves the leader peptide to produce active lantibiotics. However, the removal mechanism of leader peptide in this class lantibiotics is still not clear. [Objective] To investigate the effects of cleavage sites on the activity of peptidases domain BovT150 and SboT150 from different streptococci. [Methods] Expression vectors for the precursor peptides with mutated cleavage sites were constructed by site-directed ligase-independent mutagenesis and then the wild-type precursors(BovAm and SboAm), their mutant precursors, as well as the corresponding peptidases(BovT150 and SboT150) were expressed and purified in E. coli. The precursors were separately incubated with each peptidase in vitro and the removal efficiencies of the leader peptides were assessed by HPLC, antimicrobial activity assay and MALDI-TOF MS. [Results] Both cleavage sites GG and GA of BovAm and SboAm allowed BovT150 to retain peptidase activity, and Gly was more suitable to be processed by BovT150. Only cleavage sites GG and GA of SboAm were accessible to SboT150, which cleaved Ala more efficiently. [Conclusion] The change of amino acid residues at the cleavage sites of leader peptides affected the efficiencies of class Ⅱ lantibiotic peptidases in varying degrees.
引文
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[19] Chiu J, March PE, Lee R, et al. Site-directed, ligase-independent mutagenesis(SLIM):a single-tube methodology approaching100%efficiency in 4 h[J]. Nucleic Acids Research, 2004, 32(21):e174
[20] Lin YH, Teng KL, Huan LD, et al. Dissection of the bridging pattern of bovicin HJ50, a lantibiotic containing a characteristic disulfide bridge[J]. Microbiological Research, 2011, 166(3):146-154
[21] Zhang J, Feng YG, Teng KL, et al. Type AII lantibiotic bovicin HJ50 with a rare disulfide bond:structure, structure-activity relationships and mode of action[J]. Biochemical Journal, 2014,461(3):497-508
[22] Oman TJ, van der Donk WA. Follow the leader:the use of leader peptides to guide natural product biosynthesis[J]. Nature Chemical Biology, 2010, 6(1):9-18
[23] Chen P, Qi FX, Novak J, et al. Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II[J]. FEMS Microbiology Letters, 2001,195(2):139-144
[24] Ishii S, Yano T, Ebihara A, et al. Crystal structure of the peptidase domain of Streptococcus ComA, a bifunctional ATP-binding cassette transporter involved in the quorum-sensing pathway[J].Journal of Biological Chemistry, 2010, 285(14):10777-10785
[2] Chatterjee C, Paul M, Xie LL, et al. Biosynthesis and mode of action of lantibiotics[J]. Chemical Reviews, 2005, 105(2):633-684
[3] Willey JM, van der Donk WA. Lantibiotics:peptides of diverse structure and function[J]. Annual Review of Microbiology, 2007,61:477-501
[4] Piper C, Cotter PD, Ross RP, et al. Discovery of medically significant lantibiotics[J]. Current Drug Discovery Technologies,2009, 6(1):1-18
[5] Hasper HE, de Kruijff B, Breukink E. Assembly and stability of nisin-lipid II pores[J]. Biochemistry, 2004, 43(36):11567-11575
[6] Hasper HE, Kramer NE, Smith JL, et al. An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II[J]. Science, 2006, 313(5793):1636-1637
[7] Mcauliffe O, Ross RP, Hill C. Lantibiotics:structure, biosynthesis and mode of action[J]. FEMS Microbiology Reviews, 2001,25(3):285-308
[8] Dischinger J, Chipalu SB, Bierbaum G. Lantibiotics:promising candidates for future applications in health care[J]. International Journal of Medical Microbiology, 2014, 304(1):51-62
[9] Zhang Q, Yu Y, Vélasquez JE, et al. Evolution of lanthipeptide synthetases[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(45):18361-18366
[10] Knerr PJ, van der Donk WA. Discovery, biosynthesis, and engineering of lantipeptides[J]. Annual Review of Biochemistry,2012, 81:479-505
[11] Xie LL. Lacticin 481:in vitro reconstitution of lantibiotic synthetase activity[J]. Science, 2004, 303(5658):679-681
[12] Havarstein LS, Diep DB, Nes IF. A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export[J]. Molecular Microbiology, 1995,16(2):229-240
[13] Furgerson Ihnken LA, Chatterjee C, van der Donk WA. In vitro reconstitution and substrate specificity of a lantibiotic protease[J].Biochemistry, 2008, 47(28):7352-7363
[14] Nishie M, Shioya K, Nagao JI, et al. ATP-dependent leader peptide cleavage by NukT, a bifunctional ABC transporter, during lantibiotic biosynthesis[J]. Journal of Bioscience and Bioengineering, 2009, 108(6):460-464
[15] Xiao HJ, Chen XZ, Chen ML, et al. Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50[J].Microbiology, 2004, 150(1):103-108
[16] Barbour A, Tagg J, Abou-Zied OK, et al. New insights into the mode of action of the lantibiotic salivaricin B[J]. Scientific Reports, 2016(6):31749
[17] Sambrook J, Russell DW. Molecular Cloning:A Laboratory Manual[M]. 3rd edition. New York:Cold Spring Harbor, 2001:1595
[18] Ma HC, Gao Y, Zhao FY, et al. Individual catalytic activity of two functional domains of bovicin HJ50 synthase BovM[J]. Acta Microbiologica Sinica, 2015, 55(1):50-58(in Chinese)马宏初,高涌,赵方圆,等.羊毛硫细菌素bovicin HJ50修饰酶BovM双功能域单独催化活性鉴定[J].微生物学报, 2015,55(1):50-58
[19] Chiu J, March PE, Lee R, et al. Site-directed, ligase-independent mutagenesis(SLIM):a single-tube methodology approaching100%efficiency in 4 h[J]. Nucleic Acids Research, 2004, 32(21):e174
[20] Lin YH, Teng KL, Huan LD, et al. Dissection of the bridging pattern of bovicin HJ50, a lantibiotic containing a characteristic disulfide bridge[J]. Microbiological Research, 2011, 166(3):146-154
[21] Zhang J, Feng YG, Teng KL, et al. Type AII lantibiotic bovicin HJ50 with a rare disulfide bond:structure, structure-activity relationships and mode of action[J]. Biochemical Journal, 2014,461(3):497-508
[22] Oman TJ, van der Donk WA. Follow the leader:the use of leader peptides to guide natural product biosynthesis[J]. Nature Chemical Biology, 2010, 6(1):9-18
[23] Chen P, Qi FX, Novak J, et al. Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II[J]. FEMS Microbiology Letters, 2001,195(2):139-144
[24] Ishii S, Yano T, Ebihara A, et al. Crystal structure of the peptidase domain of Streptococcus ComA, a bifunctional ATP-binding cassette transporter involved in the quorum-sensing pathway[J].Journal of Biological Chemistry, 2010, 285(14):10777-10785
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