Institute of Biological Chemistry, Academia Sinicagrid.28665.3f, Taipei, Taiwan.
Institute of Biochemical Sciences, College of Life Science, National Taiwan Universitygrid.19188.39, Taipei, Taiwan.
mBio. 2021 Feb 22;13(1):e0364621. doi: 10.1128/mbio.03646-21. Epub 2022 Feb 15.
Bacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide -acetylglucosamine (GlcNAc) and -acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino acid composition of the peptide is unique, with l-Ala added at the first position in most bacteria but with l-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of l-Ser instead of l-Ala into peptide stems, but its mechanistic function is unknown. Here, we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor by-product UDP-MurNAc-l-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis. YfiH is a peptidoglycan (PG)-editing factor required for the maintenance of specific amino acid compositions of the stem peptides. However, the activity of YfiH has not been deciphered, and the editing mechanism involving YfiH has remained a mystery. Through X-ray crystallographic and biochemical analyses, we demonstrate that YfiH is a hydrolase with a previously unknown activity specific for the UDP-MurNAc-monopeptide, one of the nucleotide precursors from the cytoplasmic steps of the PG biosynthesis pathway. YfiH selectively hydrolyzes UDP-MurNAc-Ser, an incorrect by-product of the biosynthesis reaction, to ensure that only the correct PG precursor, UDP-MurNAc-Ala, is incorporated. Therefore, this work reveals coupled synthetic and editing reactions in the cytoplasmic steps of PG biosynthesis.
细菌细胞被包裹在肽聚糖（PG）中，肽聚糖是由二糖-N-乙酰葡萄糖胺（GlcNAc）和-N-乙酰胞壁酸（MurNAc）通过肽主干交联而成的聚合物。PG 在细胞质中作为 UDP-MurNAc-肽前体合成，其中肽的氨基酸组成是独特的，大多数细菌中的第一个位置添加的是 l-Ala，但在一些细菌中添加的是 l-Ser 或 Gly。YfiH 是一种 PG 编辑因子，其缺失会导致 l-Ser 而不是 l-Ala 错误掺入肽主干，但它的机制功能尚不清楚。在这里，我们报告了结合底物和产物的 YfiH 的晶体结构，表明 YfiH 是一种细胞质酰胺酶，通过优先切割核苷酸前体的副产物 UDP-MurNAc-l-Ser，控制正确氨基酸掺入核苷酸前体。这项工作揭示了肽聚糖生物合成细胞质步骤中的一种编辑机制。 YfiH 是一种肽聚糖（PG）编辑因子，对于维持肽主干的特定氨基酸组成是必需的。然而，YfiH 的活性尚未被破解，涉及 YfiH 的编辑机制仍然是一个谜。通过 X 射线晶体学和生化分析，我们证明 YfiH 是一种具有以前未知活性的水解酶，专门针对 UDP-MurNAc-单肽，即 PG 生物合成途径细胞质步骤中的核苷酸前体之一。YfiH 选择性地水解 UDP-MurNAc-Ser，这是生物合成反应的一个不正确的副产物，以确保只有正确的 PG 前体 UDP-MurNAc-Ala 被掺入。因此，这项工作揭示了 PG 生物合成细胞质步骤中的耦合合成和编辑反应。
