A Novel Epimerase Catalyzing Multiple Isomerization of Amino Acid Residues of Ribosomal Peptide

• Material Type: 博士論文
• Title: A Novel Epimerase Catalyzing Multiple Isomerization of Amino Acid Residues of Ribosomal Peptide
• Author/Editor: Xiao, Wanlu
• Author Heading: Xiao, Wanlu
• Publication, Distribution, etc.: Hokkaido University
• Publication Date: 2023-09-25
• Publication Date (W3CDTF): 2023-09-25
• Alternative Title: リボソームペプチドの複数アミノ酸残基を異性化する新規エピメラーゼ
• Degree grantor/type: 北海道大学
• Date Granted: 2023-09-25
• Date Granted (W3CDTF): 2023-09-25
• Dissertation Number: 甲第15640号
• Degree Type: 博士(工学)
• Text Language Code: eng
• NDC: 500
• Target Audience: 一般
• Note (General): Salinipeptins, grisemycin, and cypemycin are ribosomally synthesized and post-translationally modified peptides (RiPPs). Among these, salinipeptins were reported to comprise 22 amino acid residues with multiple D-amino acids, and its biosynthetic gene cluster was identified. However, no genes homologous to known isomerases such as epimerases and racemases existed in the cluster, but a gene, sinL, which showed no similarities to function known enzymes, located in the cluster, suggesting that SinL might be a novel epimerase. Actually, biosynthetic gene clusters of grisemycin and cypemycin also possess orthologs of sinL, although the chirality of amino acids composing grisemycin and cypemycin has not been reported. Therefore, I first examined grisemycin also contains D amino acid residues. By heterologous expression of grisemycin biosynthetic gene cluster (grm) in Streptomyces lividans, grisemycin was confirmed to contain multiple D-amino acids, in a similar manner to salinipeptins. The heterologous expression experiments also confirmed the involvement of a novel peptide epimerase in grisemycin biosynthesis. Gene-deletion experiments indicated that grmL, an ortholog of sinL, was indispensable for grisemycin production and that the epimerization preceded decarboxylation and methylation, which are other modifications installed into the precursor peptides of grisemycin (GrmA).To obtain further evidence that GrmL encodes the novel epimerase, recombinant precursor peptide (GrmA) and GrmL were prepared and used for in vitro analysis. However, no isomerase activity was observed under various conditions. Considering that grisemycin contains dehydroamino acids and its putative biosynthetic gene, grmH, exists in the gene cluster, the dehydration reaction might occur before isomerization. To examine the possibility, recombinant GrmH was prepared and incubated with GrmA, but no dehydration activity was detected. Because GrmL is the novel enzyme found in the Streptomyces strain, I considered the possibility that GrmL might require a cofactor specifically utilized in the Streptomyces strain. I therefore employed the above-mentioned heterologous expression system again. When grmA, grmH, and grmL were co-expressed, a dehydrated and isomerized GrmA was produced, but grmA and either of the two resulted in the production of no modified GrmA. The results suggested that both GrmH and GrmL, which were shown to constitute a protein complex by a pulldown assay, were required to catalyze the dehydration, epimerization, and proteolytic cleavage of a precursor peptide GrmA by in vivo experiments.; (主査) 教授 松本 謙一郎, 教授 渡慶次 学, 教授 大利 徹, 准教授 小笠原 泰志, 准教授 南 篤志; 総合化学院（総合化学専攻）
• DOI: 10.14943/doctoral.k15640; https://doi.org/10.14943/doctoral.k15640
• Persistent ID (NDL): info:ndljp/pid/13122813; https://dl.ndl.go.jp/pid/13122813
• Collection: 障害者向け資料
• Collection (Materials For Handicapped People:1): テキストデータ
• Collection (particular): 国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文; https://dl.ndl.go.jp/collections/A00014
• Acquisition Basis: 博士論文（自動収集）
• Date Accepted (W3CDTF): 2023-12-15T22:13:26+09:00
• Format (IMT): application/pdf
• Access Restrictions: 国立国会図書館内限定公開
• Service for the Digitized Contents Transmission Service: 図書館・個人送信対象外
• Availability of remote photoduplication service: 可
• Periodical Title (URI): http://dx.doi.org/10.14943/doctoral.k15640; http://hdl.handle.net/2115/90816
• Data Provider (Database): 国立国会図書館 : 国立国会図書館デジタルコレクション; https://dl.ndl.go.jp