HUSCAP logo Hokkaido Univ. logo

Hokkaido University Collection of Scholarly and Academic Papers >
Theses >
博士 (工学) >

Studies on New Enzymes Responsible for Peptidoglycan Biosynthesis

Files in This Item:
Ruoyin_Feng.pdf18.99 MBPDFView/Open
Please use this identifier to cite or link to this item:http://doi.org/10.14943/doctoral.k13812
Related Items in HUSCAP:

Title: Studies on New Enzymes Responsible for Peptidoglycan Biosynthesis
Other Titles: ペプチドグリカンの生合成に関与する新規酵素に関する研究
Authors: 馮, 若茵1 Browse this author
Authors(alt): Feng, Ruoyin1
Issue Date: 25-Sep-2019
Abstract: Bacterial cell walls contain D-glutamate (Glu) as a component of peptidoglycans. In general, D-Glu is synthesized from L-Glu by Glu racemase (MurI, EC 5.1.1.3). In some bacteria, D-amino acid aminotransferase (EC 2.6.1.21) supplies D-Glu by transamination from D-alanine (Ala) to α-ketoglutarate. However, in my laboratory, detailed bioinformatic analysis was performed and it was revealed that Xanthomonas oryzae has no orthologs of both of the genes in spite of the fact that X. oryzae is prototroph for D-Glu. This fact strongly suggested that the bacterium would have an alternative D-Glu biosynthetic pathway. In this study, I explored and analyzed the novel enzymes responsible for D-Glu biosynthesis. In chapter 2, I performed shotgun-cloning experiments with a D-Glu auxotrophic Escherichia coli mutant as host and X. oryzae as DNA donor. I obtained two complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-L-Ala-D-Glu synthetase), respectively. By in vitro experiment with recombinant enzymes, I revealed that XOO_1320 is an enzyme to ligate L-Glu to UDP-MurNAc-L-Ala for the first example of MurD utilizing L-Glu. XOO_1319 is a novel enzyme catalyzing epimerization of the terminal L-Glu of the product in the presence of ATP and Mg2+. XOO_1319 and XOO_1320 were renamed as MurL and MurD2, respectively. In chapter 3, to study the isomer recognition mechanism of MurD and MurD2, the structure models of MurD2 were constructed based on E. coli MurD (MurDec) structure by docking simulations. Several amino acid residues, which were probably responsible for L-Glu recognition, were then replaced with corresponding amino acid residues in MurDec. Consequently, I obtained a mutated MurD2 enzyme accepting only D-Glu as the substrate by two amino acid substitutions. I also tried to convert the iii substrate specificity of MurDec by the same strategy, but the mutated enzymes still accepted only D-Glu. Next, I used another MurD from Streptococcus mutans (MurDsm) for random screenings of mutant enzymes accepting L-Glu. Consequently, I obtained a mutated MurDsm that had one amino acid substitution and slightly accepted L-Glu. A mutated MurDec possessing the corresponding one amino acid substitution also accepted L-Glu. Chapter 4 is the summary of the study.
Conffering University: 北海道大学
Degree Report Number: 甲第13812号
Degree Level: 博士
Degree Discipline: 工学
Examination Committee Members: (主査) 教授 松本 謙一郎, 教授 髙木 睦, 准教授 南 篤志, 教授 大利 徹
Degree Affiliation: 総合化学院(総合化学専攻)
Type: theses (doctoral)
URI: http://hdl.handle.net/2115/75882
Appears in Collections:課程博士 (Doctorate by way of Advanced Course) > 総合化学院(Graduate School of Chemical Sciences and Engineering)
学位論文 (Theses) > 博士 (工学)

Export metadata:

OAI-PMH ( junii2 , jpcoar )

MathJax is now OFF:


 

Feedback - Hokkaido University