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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92509
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dc.contributor.advisor張崇毅zh_TW
dc.contributor.advisorChung-I Changen
dc.contributor.author李孟昇zh_TW
dc.contributor.authorMeng-Sheng Leeen
dc.date.accessioned2024-03-26T16:23:04Z-
dc.date.available2024-03-27-
dc.date.copyright2024-03-26-
dc.date.issued2024-
dc.date.submitted2024-02-27-
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92509-
dc.description.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。
zh_TW
dc.description.abstractPeptidoglycan (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.
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dc.description.tableofcontents國立臺灣大學博士學位論文口試委員會審定書 I
國立臺灣大學學位論文學術倫理暨原創性比對聲明書 II
誌謝 III
中文摘要 IV
英文摘要 V
目次 VI
Table of Contents VII
List of Tables IX
List of Figures X
List of Abbreviations XII

Chapter 1. Introduction 1
1.1 Cell envelope 1
1.2 Peptidoglycan and muropeptide 1
1.3 Biosynthesis of muropeptide 4
1.3.1 The peptide stem biosynthetic pathway 5
1.3.2 The Mur ligases 6
1.3.3 The specificity of Mur ligases 8
1.3.4 The effects of muropeptide misincorporation on bacteria 10
1.4 Peptidoglycan-editing factor (YfiH; PgeF) 10
1.5 Motivation of research 12
Chapter 2. Materials and Methods 15
2.1 Cloning and mutagenesis 15
2.2 Protein expression and purification 15
2.3 Crystallization, structure determination, and refinement 16
2.4 Synthesis of UDP-MurNAc-peptide derivatives 18
2.5 Synthesis of MurNAc-L-Ser derivatives 18
2.6 Enzyme activity assay 20
Chapter 3. Results 21
3.1 Overall structure of YfiH-C107A bound to UDP-MurNAc 21
3.2 Interaction of YfiH with UDP-MurNAc-monopeptide 23
3.3 UDP-MurNAc-L-Ser is the preferred substrate of YfiH 24
3.4 The substrate specificity of YfiH is determined by Gln69 27
3.5 The amidase activity of YfiH for UDP-MurNAc-Gly 28
3.6 The prokaryotic YfiH exhibits high conservation in the C-terminal domain of human FAMIN/Lacc1 28
Chapter 4. Discussion 31
4.1 The peptidoglycan proofreading role of YfiH is specific for MurC ligase 31
4.2 Metal ion is dispensable for the PG-precursor hydrolysis activity of YfiH 32
4.3 YfiH may be involved in Mre complex 33
4.4 The function of PG recognition may exist in other DUF152 proteins 35
4.5 YfiH as a druggable target for synergistic antibiotics development 36
Figures 38
Tables 57
Appendices 67
References 74
List of Publications 81
Reprint Permissions/Approvals 82
-
dc.language.isoen-
dc.subject乙酰氨基葡萄糖zh_TW
dc.subject胜肽聚醣zh_TW
dc.subjectYfiH蛋白zh_TW
dc.subject脫醯胺水解酶zh_TW
dc.subject乙酰胞壁酸zh_TW
dc.subjectX光繞射zh_TW
dc.subjectUDP-MurNAc-L-Seren
dc.subjectYfiHen
dc.subjectPeptidoglycanen
dc.subjectN-acetyl-glucosamineen
dc.subjectN-acetyl-muramic aciden
dc.subjectAmidaseen
dc.subjectX-ray protein crystallographyen
dc.title以結構及活性分析方法來解析YfiH的肽聚醣編輯機制zh_TW
dc.titleUnderstanding the peptidoglycan-editing mechanism of YfiH by structural and functional analysesen
dc.typeThesis-
dc.date.schoolyear112-2-
dc.description.degree博士-
dc.contributor.oralexamcommittee侯明宏;曾秀如;翁瑞霞;何孟儒zh_TW
dc.contributor.oralexamcommitteeMing-Hon Hou;Shiou-Yu Tzeng;Jui-Hsia Weng;Meng-Ru Hoen
dc.subject.keywordYfiH蛋白,胜肽聚醣,乙酰氨基葡萄糖,乙酰胞壁酸,脫醯胺水解酶,X光繞射,zh_TW
dc.subject.keywordYfiH,Peptidoglycan,N-acetyl-glucosamine,N-acetyl-muramic acid,Amidase,X-ray protein crystallography,UDP-MurNAc-L-Ser,en
dc.relation.page85-
dc.identifier.doi10.6342/NTU202400754-
dc.rights.note同意授權(限校園內公開)-
dc.date.accepted2024-02-27-
dc.contributor.author-college生命科學院-
dc.contributor.author-dept生化科學研究所-
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