Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis
Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis
复制标题
脂肽生物合成中非核糖体肽合成酶起始缩合结构域脂肪酰基底物选择性的探索和改造
DOI：
10.1002/biot.201900175
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发表时间：
2019
影响因子：
4.7
通讯作者：
Feng Yan
中科院分区：
文献类型：
--
作者：
Liu Qian;Fan Wenjie;Zhao Yajin;Deng Zixin;Feng Yan
脂肽由非核糖体肽合成酶（NRPS）产生，含有多种脂肪酰基部分，这些部分是抗生素效力的主要决定因素。脂质链通过脂质引发并入肽基主链，该过程包括游离脂肪酸活化和随后的起始剂缩合结构域（C1）催化的脂肪酰基部分与氨酰基底物的缀合。因此，对脂质起始生物催化剂的透彻理解将显着扩大其生产新型抗生素的潜力。在这里，生物化学测定，计算机分析，和诱变研究，用于最终确定特定的氨基酸残基，控制脂肽A54145中的C1的脂肪酰基底物选择性。计算机对接研究已经确定了四个候选氨基酸，随后的体外试验证实了它们对控制底物选择性的通道的功能贡献。两个工程改造的变体与单点突变C1被发现改变对非天然脂肪酰基底物的底物选择性。从本研究中获得的C1催化贡献的详细机理见解将有助于未来NPRS生物催化剂的努力
Lipopeptides are produced by nonribosomal peptide synthetases (NRPSs) and contain diverse fatty acyl moieties that are major determinants of antibiotic potency. The lipid chains are incorporated into peptidyl backbones via lipoinitiation, a process comprising free fatty acid activation and the subsequent starter condensation domain (C1)‐catalyzed conjugation of fatty acyl moieties onto the aminoacyl substrates. Thus, a thorough understanding of lipoinitiation biocatalysts would significantly expand their potential to produce novel antibiotics. Here, biochemical assays, in silico analysis, and mutagenesis studies are used to ultimately identify the specific amino acid residues that control the fatty acyl substrate selectivity of C1 in lipopeptide A54145. In silico docking study has identified four candidate amino acids, and subsequent in vitro assays confirmed their functional contribution to the channel that controls substrate selectivity. Two engineered variants with single point mutations in C1 are found to alter the substrate selectivity toward nonnatural fatty acyl substrates. The detailed mechanistic insights into the catalytic contribution of C1 obtained from the present study will facilitate future NPRS biocatalyst efforts