Department of Biotechnology, Graduate School of Agricultural and Life science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Angew Chem Int Ed Engl. 2021 Jun 21;60(26):14554-14562. doi: 10.1002/anie.202102760. Epub 2021 May 17.
Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and α-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.
非核糖体肽合成酶（NRPSs）是生物工程中生成有用肽的有吸引力的靶点。FmoA3 是一个由杂环化（Cy）、腺苷酸化（A）和肽酰载体蛋白（PCP）结构域组成的单一模块化 NRPS。它使用α-甲基-l-丝氨酸合成 4-甲基恶唑啉环，可能在前一个模块 FmoA2 中的另一个 Cy 结构域中完成。在这里，我们通过 X 射线晶体学（apo 形式，带有腺嘌呤二核苷酸和α-甲基-l-丝氨酰-AMP）和低温电子显微镜单颗粒分析确定了 FmoA3 的头到尾同源二聚体结构，并进行了定点突变实验。数据表明，由于 Ala688 周围的额外空间，α-甲基-l-丝氨酸可以容纳在活性位点中。FmoA2 和 FmoA3 的 Cy 结构域分别催化肽键形成和杂环化。FmoA3 的 Cy 结构域似乎失去了其供体 PCP 结合活性。综合数据支持了 FmoA3 的拟议催化循环。
