非核糖体肽合成酶基因腺苷酰化结构域序列克隆及分析
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摘要
微生物许多非核糖体肽类次生代谢产物主要是由非核糖体肽合成酶(NRPS)催化合成。参考Gontang发布的非核糖体肽合成酶(NRPS)通用引物设计扩增NRPS腺苷酰化结构域基因序列的特异引物,从海洋链霉菌L1的基因组DNA中扩增获得一个715 bp的NRPS基因序列。测序结果及比对分析表明该片段属于NRPS腺苷酰化结构域部分序列。对其拟翻译的氨基酸序列组成成分、理化性质进行分析,显示其包含AFD class I超基因家族核心结合区,为NRPS腺苷酰化结构域(A结构域)所在区域。对氨基酸序列的二级结构预测和三级结构模拟,发现与数据库中肠菌素合酶F组分的结构相似。为后续研究A结构域的特异性及完整NRPS基因簇克隆提供了参考。
Many microbial non-ribosomal peptide secondary metabolites are mainly synthesized catalytically by nonribosomal peptide synthetase(NRPS). Reffered to the NRPS universal primers released by Gontang, one pair of specific primers were designed to amplify NRPS gene fragment from marine Streptomyces globisporus L1 genomic DNA and obtained a 715 bp of NRPS gene sequence. Sequencing results and comparison analysis showed that the fragment belonged to part sequences of NRPS adenylation domain. The composition, physical and chemical properties of the corresponding amino acid sequences planed to be translated were analyzed, and showed that it contained one AFD class I superfamily core binding region to be where the NRPS adenylation structural domain(A structural domain) were. Through secondary structure prediction of amino acid sequence and simulation of tertiary structure, it was found that they were similar to that of enterobactin synthase component F in database. These had provided theoretical basis on domain specificity and the cloning of complete NRPS gene clusters in the follow-up study on A structural domain.
Many microbial non-ribosomal peptide secondary metabolites are mainly synthesized catalytically by nonribosomal peptide synthetase(NRPS). Reffered to the NRPS universal primers released by Gontang, one pair of specific primers were designed to amplify NRPS gene fragment from marine Streptomyces globisporus L1 genomic DNA and obtained a 715 bp of NRPS gene sequence. Sequencing results and comparison analysis showed that the fragment belonged to part sequences of NRPS adenylation domain. The composition, physical and chemical properties of the corresponding amino acid sequences planed to be translated were analyzed, and showed that it contained one AFD class I superfamily core binding region to be where the NRPS adenylation structural domain(A structural domain) were. Through secondary structure prediction of amino acid sequence and simulation of tertiary structure, it was found that they were similar to that of enterobactin synthase component F in database. These had provided theoretical basis on domain specificity and the cloning of complete NRPS gene clusters in the follow-up study on A structural domain.
引文
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[11] Gontang EA, Gaudêncio SP, Fenical W, et al. Sequence-based analysis of secondary-metabolite biosynthesis in marine actinobacteria[J]. Applied and Environmental Microbiology, 2010, 76(8):2487-2499.
[12] Conti E, Stachelhaus T, Marahiel MA, et al. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S[J]. EMBO Journal, 1997,16(14):4171-4183.
[13] Yonus H, Neumann P, Zimmermann S, et al. Cystal structure of DltA, Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains[J]. Journal of Biological Chemistry, 2008,283(47):32484-32491.
[14] Tanovic A, Samel SA, Essen LO, et al. Crystal structure of the termination module of a nonribosomal peptide synthetase[J]. Science, 2008,321(5889):659-660.
[15] Miller BR, Sundlov JA, Drake E, et al. Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases[J]. Proteins Structure Function and Bioinformatics, 2014,82(10):2691-2702.
[16] Kittil? T, Schoppet M, Cryle MJ, Online pyrophosphate assay for analyzing adenylation domains of non-ribosomal peptide synthetases[J]. Chembiochem, 2016, 17(7):576-584.
[17] Henderson JC, Fage CD, Cannon JR, et al. Antimicrobial peptide resistance of vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases[J]. ACS Chemical Biology, 2014, 9(10):2382-2392.
[18] Kittil? T, Mollo A, Charkoudian LK, et al. New structural data reveal the motion of carrier proteins in nonribosomal peptide synthesis[J]. Angew. Chem. Int. Ed. Engl., 2016,55(34):9834-9840.
[2] 郑宗明, 顾晓波, 俞海清,等. 非核糖体肽合成酶主要结构域的研究进展[J]. 中国抗生素杂志, 2005,30(2):120-124.
[3] Roderich DS,Andi M. Nonribosomal peptide synthesis-principles and prospects[J]. Angewandte Chemie International Edition,2017,56(14):3770-3821
[4] 李红玲. 非核糖体肽合成酶结构研究进展[J]. 临床合理用药, 2013, 6(10):180-181.
[5] Labby KJ, Watsula SG, Garneau-Tsodikova S. Interrupted adenylation domains: unique bifunctional enzyme involved in nonribosomal peptide biosynthesis[J]. Natural product Report, 2015, 32(5):641-653.
[6] Nielsen ML, Isbrandt T, Petersen LM, et al. Linker flexibility facilitates module exchange in fungal hybrid PKS-NRPS engineering[J]. PLoS ONE, 2016, 11(8):1-18.
[7] Hansen FT, Gardiner DM, Lys?e E, et al. An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in fusarium[J]. Fungal Genetics and Biology, 2015,75: 20-29.
[8] Konno S, Ishikawa F, Suzuki T, et al. Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases[J]. Chemical Communications, 2015, 51(12):2262-2265.
[9] Weber T, Rausch C, Lopez P, et al. CLUSEAN: a computer-based framework for the automated analysis of bacterial secondary metabolite biosynthetic gene clusters[J]. Journal of Biotechnol, 2009, 140(1-2):13-17.
[10] 李根, 薛永常. 非核糖体肽合成酶缩合结构域基因片段克隆[J]. 工业微生物, 2017, 47(2):37-42.
[11] Gontang EA, Gaudêncio SP, Fenical W, et al. Sequence-based analysis of secondary-metabolite biosynthesis in marine actinobacteria[J]. Applied and Environmental Microbiology, 2010, 76(8):2487-2499.
[12] Conti E, Stachelhaus T, Marahiel MA, et al. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S[J]. EMBO Journal, 1997,16(14):4171-4183.
[13] Yonus H, Neumann P, Zimmermann S, et al. Cystal structure of DltA, Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains[J]. Journal of Biological Chemistry, 2008,283(47):32484-32491.
[14] Tanovic A, Samel SA, Essen LO, et al. Crystal structure of the termination module of a nonribosomal peptide synthetase[J]. Science, 2008,321(5889):659-660.
[15] Miller BR, Sundlov JA, Drake E, et al. Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases[J]. Proteins Structure Function and Bioinformatics, 2014,82(10):2691-2702.
[16] Kittil? T, Schoppet M, Cryle MJ, Online pyrophosphate assay for analyzing adenylation domains of non-ribosomal peptide synthetases[J]. Chembiochem, 2016, 17(7):576-584.
[17] Henderson JC, Fage CD, Cannon JR, et al. Antimicrobial peptide resistance of vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases[J]. ACS Chemical Biology, 2014, 9(10):2382-2392.
[18] Kittil? T, Mollo A, Charkoudian LK, et al. New structural data reveal the motion of carrier proteins in nonribosomal peptide synthesis[J]. Angew. Chem. Int. Ed. Engl., 2016,55(34):9834-9840.