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A Novel Epimerase Catalyzing Multiple Isomerization of Amino Acid Residues of Ribosomal Peptide
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| Title: | A Novel Epimerase Catalyzing Multiple Isomerization of Amino Acid Residues of Ribosomal Peptide |
| Other Titles: | 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 |
| Authors: | Xiao, Wanlu Browse this author |
| Issue Date: | 25-Sep-2023 |
| Publisher: | Hokkaido University |
| Abstract: | 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. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 甲第15640号 |
| Degree Level: | 博士 |
| Degree Discipline: | 工学 |
| Examination Committee Members: | (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 |
| Degree Affiliation: | 総合化学院（総合化学専攻） |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/90816 |
| 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）
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