{"title":"SIRT5 突变体揭示了 NAD+ 结合袋中保守的天冬酰胺和谷氨酰胺残基的作用","authors":"Takeshi Yokoyama, Yuki Takayama, Mineyuki Mizuguchi, Yuko Nabeshima, Katsuhiro Kusaka","doi":"10.1002/1873-3468.14961","DOIUrl":null,"url":null,"abstract":"<p>SIRT5, one of the mammalian sirtuins, specifically recognizes succinyl-lysine residues on proteins and catalyzes the desuccinylation reaction. In this study, we characterized SIRT5 mutants with hydrophobic amino acid substitutions at Q140 and N141, in addition to the catalytic residue H158, known as an active site residue, by the Michaelis–Menten analysis and X-ray crystallography. Kinetic analysis showed that the catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) of the Q140L and N141V mutants decreased to 0.02 times and 0.0038 times that of the wild-type SIRT5, respectively, with the activity of the N141V mutant becoming comparable to that of the H158M mutant. Our findings indicate that N141 contributes significantly to the desuccinylation reaction.</p>","PeriodicalId":12142,"journal":{"name":"FEBS Letters","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"SIRT5 mutants reveal the role of conserved asparagine and glutamine residues in the NAD+-binding pocket\",\"authors\":\"Takeshi Yokoyama, Yuki Takayama, Mineyuki Mizuguchi, Yuko Nabeshima, Katsuhiro Kusaka\",\"doi\":\"10.1002/1873-3468.14961\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>SIRT5, one of the mammalian sirtuins, specifically recognizes succinyl-lysine residues on proteins and catalyzes the desuccinylation reaction. In this study, we characterized SIRT5 mutants with hydrophobic amino acid substitutions at Q140 and N141, in addition to the catalytic residue H158, known as an active site residue, by the Michaelis–Menten analysis and X-ray crystallography. Kinetic analysis showed that the catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) of the Q140L and N141V mutants decreased to 0.02 times and 0.0038 times that of the wild-type SIRT5, respectively, with the activity of the N141V mutant becoming comparable to that of the H158M mutant. Our findings indicate that N141 contributes significantly to the desuccinylation reaction.</p>\",\"PeriodicalId\":12142,\"journal\":{\"name\":\"FEBS Letters\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-06-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"FEBS Letters\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/1873-3468.14961\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"FEBS Letters","FirstCategoryId":"99","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/1873-3468.14961","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
SIRT5 mutants reveal the role of conserved asparagine and glutamine residues in the NAD+-binding pocket
SIRT5, one of the mammalian sirtuins, specifically recognizes succinyl-lysine residues on proteins and catalyzes the desuccinylation reaction. In this study, we characterized SIRT5 mutants with hydrophobic amino acid substitutions at Q140 and N141, in addition to the catalytic residue H158, known as an active site residue, by the Michaelis–Menten analysis and X-ray crystallography. Kinetic analysis showed that the catalytic efficiency (kcat/Km) of the Q140L and N141V mutants decreased to 0.02 times and 0.0038 times that of the wild-type SIRT5, respectively, with the activity of the N141V mutant becoming comparable to that of the H158M mutant. Our findings indicate that N141 contributes significantly to the desuccinylation reaction.
期刊介绍：
FEBS Letters is one of the world''s leading journals in molecular biology and is renowned both for its quality of content and speed of production. Bringing together the most important developments in the molecular biosciences, FEBS Letters provides an international forum for Minireviews, Research Letters and Hypotheses that merit urgent publication.
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