以結構及活性分析方法來解析YfiH的肽聚醣編輯機制

Abstract

細菌細胞壁以多層的胜肽聚醣 (peptidoglycan)交疊而成，主要由乙酰化的氨基葡萄糖 (GlcNAc; NAG)、乙酰胞壁酸 (MurNAc; NAM)與一段保守的短鏈胜肽所建構。由於胜肽聚醣的結構與成分在原核生物間高度保守，暗示了細菌可能存在著維持細胞壁結構的校對機制。另一方面，近期研究也發現YfiH蛋白有助於維持大腸桿菌中細胞壁組成的正確性，但其詳細的分子機轉仍有待釐清。

本論文旨在通過結構生物學方法闡明細菌YfiH蛋白的肽聚醣校對機制。透過X光繞射解析失活突變型YfiH-C107A的結構，本研究發現該蛋白會與大腸桿菌生長時被錯誤修飾的胜肽聚醣前驅物: UDP-MurNAc-L-Ser (UMS)結合。本論文進一步證明YfiH具有脫醯胺水解酶 (amidase)的活性，能選擇性將錯誤引入的單胜肽從UMS上的UDP-MurNAc上移除。而此酵素能直接與前驅物的胜肽位置作用的胺基酸中，其第69號麩胺酸 (Glutamine; Gln)，該殘基對於水解受質的選擇性扮演了關鍵性的角色。這些證據表明YfiH是一種位於細胞質的酵素，通過移除不正確的胜肽聚醣前驅物來維持細菌細胞壁組成的正確性。相較過去的研究多專注於成熟細胞壁如何被修復，本研究發現原核生物也存在防範錯誤的前驅物進入細胞壁中的機制。

Peptidoglycan (PG) stands as a crucial element within the bacterial cell wall, consisting of N-acetyl-glucosamine (GlcNAc; NAG), N-acetyl-muramic acid (MurNAc; NAM), and peptide stems. These units form a mesh-like structure through glycosidic bonds and interpeptide linkages. Peptidoglycan biosynthesis initiates with cytoplasmic UDP-MurNAc-peptide precursors, featuring a conserved peptide stem composition, commonly incorporating L-alanine in the first position in most bacteria. Recent studies highlight the crucial role of YfiH in maintaining a specific amino acid composition in PG, although the exact maintenance mechanism remains unclear.

The dissertation aims to elucidate the peptidoglycan-correcting role of YfiH using structural biological approaches. X-ray protein crystallography revealed that the catalytically inactive E. coli YfiH mutein, YfiH-C107A, can bind to an endogenously trapped UDP-MurNAc, as well as the non-canonical muropeptide precursor UDP-MurNAc-L-Ser (UMS). Moreover, YfiH preferentially cleaves UDP-MurNAc-L-Ser (UMS) to UDP-MurNAc, serving as a cytoplasmic amidase. This enzymatic activity plays a crucial role in ensuring the precision of amino acid incorporation into nucleotide precursors by eliminating by-products catalyzed by MurC. In addition, the YfiH Gln69 residue, directly interacting with the monopeptide moiety of nucleotide precursors, is essential for selectively hydrolyzing UMS. This study proposes a previously unknown proofreading mechanism in the cytoplasmic stages of peptidoglycan biosynthesis, wherein YfiH selectively hydrolyzes the incorrect precursor, ensuring the accuracy of the amino acid composition in PG.