Characterization of Pyridomycin B Reveals the Formation of Functional Groups in Antimycobacterial Pyridomycin
Characterization of Pyridomycin B Reveals the Formation of Functional Groups in Antimycobacterial Pyridomycin
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DOI：
10.1128/aem.02035-21
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发表时间：
2022-03-01
影响因子：
4.4
通讯作者：
Lin，Shuangjun
中科院分区：
文献类型：
--
作者：
Huang，Tingting;Zhou，Zihua;Lin，Shuangjun
吡多霉素是一种具有强效抗分枝杆菌活性的环缩肽，特异性抑制结核分枝杆菌的InhA烯酰还原酶。构效关系研究表明，吡多霉素核心系统中的烯醇酸部分是一个重要的药效基团，C-10位羟基的天然构型对吡多霉素的生物活性有重要影响。吡多霉素的环状结构是由非核糖体肽合成酶（NRPS）和聚酮合成酶（PKS）杂交系统（PyrE-PyrF-PyrG）产生的。生物信息学分析表明，短链脱氢酶/还原酶（SDR）家族蛋白Pyr 2在吡啶霉素途径中充当3-氧代酰基酰基载体蛋白（ACP）还原酶。pyr 2的失活导致吡多霉素B的积累，吡多霉素B是一种新的吡多霉素类似物，其特征在于吡啶丙氨酸部分具有烯醇部分和饱和的3-甲基戊酸基团。吡多霉素B的结构表明，当PKS-NRPS组装的中间体仍然以特殊的节能方式连接到最后的NRPS模块时，Pyr 2催化酮还原以形成吡啶基丙氨酸部分中的C-10羟基和衍生自异亮氨酸的烯醇酸部分的双键形成，而不是作为后剪裁酶。Ser-His-LyS残基构成Pyr 2的活性位点，这与大多数SDR中典型保守的基于Tyr的催化三联体不同。定点突变表明活性位点的His 154是吡啶霉素B生产的关键残基。这些发现将提高我们对吡多霉素生物合成逻辑的理解，确定吡多霉素中烯醇酯双键形成的缺失环节，并使吡多霉素衍生物的化学多样性得以创造。重要信息结核病（TB）是世界上主要的感染致死原因之一。近年来，作为抗结核天然产物的吡多霉素因被确定为结核分枝杆菌InhA烯酰还原酶的靶向抑制剂而受到广泛关注。在这项研究中，我们报告了一个新的吡多霉素类似物从突变体HTT 12，证明了一个以前被忽视的genepyr 2在吡多霉素的生物合成途径中的重要作用，并暗示Pyr 2功能作为一种独特的催化机制，有助于形成吡多霉素的功能基团。由于烯醇部分对吡多霉素的药物活性很重要，我们的工作将扩大我们对SDR家族蛋白质作用机制的理解，并为未来新的吡多霉素衍生物的生物工程奠定基础。
Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacterium tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in the pyridomycin core system is an important pharmacophoric group, and the natural configuration of the C-10 hydroxyl contributes to the bioactivity of pyridomycin. The ring structure of pyridomycin was generated by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) hybrid system (PyrE-PyrF-PyrG). Bioinformatics analysis reveals that short-chain dehydrogenase/reductase (SDR) family protein Pyr2 functions as a 3-oxoacyl acyl carrier protein (ACP) reductase in the pyridomycin pathway. Inactivation ofpyr2resulted in accumulation of pyridomycin B, a new pyridomycin analogue featured with enol moiety in pyridyl alanine moiety and a saturated 3-methylvaleric acid group. The elucidated structure of pyridomycin B suggests that rather than functioning as a post-tailoring enzyme, Pyr2 catalyzes ketoreduction to form the C-10 hydroxyl group in pyridyl alanine moiety and the double bond formation of the enolic acid moiety derived from isoleucine when the intermediate assembled by PKS-NRPS machinery is still tethered to the last NRPS module in a special energy-saving manner. Ser-His-Lys residues constitute the active site of Pyr2, which is different from the typically conserved Tyr-based catalytic triad in the majority of SDRs. Site-directed mutation identified that His154 in the active site is a critical residue for pyridomycin B production. These findings will improve our understanding of pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin, and enable the creation of chemical diversity of pyridomycin derivatives.IMPORTANCETuberculosis (TB) is one of the world’s leading causes of death by infection. Recently, pyridomycin, the antituberculous natural product fromStreptomyceshas garnered considerable attention for being determined as a target inhibitor of InhA enoyl reductase of Mycobacterium tuberculosis. In this study, we report a new pyridomycin analogue from mutant HTT12, demonstrate the essential role of a previously ignored genepyr2in pyridomycin biosynthetic pathway, and imply that Pyr2 functions as atransketoreductase (KR) contributing to the formation of functional groups of pyridomycin utilizing a distinct catalytic mechanism. As enol moiety are important for pharmaceutical activities of pyridomycin, our work would expand our understanding of the mechanism of SDR family proteins and set the stage for future bioengineering of new pyridomycin derivatives.