{"title":"Structural and functional analysis of Bacillus sarcosine oxidase and its activity toward cyclic imino acids.","authors":"Yuqi Zhang, Yoshitaka Nakajima, Masae Kurobe, Tsutomu Nakamura, Tomoki Himiyama, Yoshiaki Nishiya","doi":"10.1002/2211-5463.70119","DOIUrl":null,"url":null,"abstract":"<p><p>This study investigated the reactivity of sarcosine oxidase (Sox) toward minor substrates through kinetic and structural analyses, along with mutational engineering to elucidate their reaction mechanisms. Sarcosine oxidase from Bacillus sp. (SoxB) recognizes the cyclic imino acids l-proline (l-Pro), d-proline (d-Pro), and l-thioproline (l-Tpr) as minor substrates. The reaction behavior varied depending on the substrates; notably, the absorption spectrum of l-Tpr exhibited charge transfer, which was characteristic of substrate inhibition. Crystal structures of the enzyme-substrate complexes suggested that Tyr254 causes spatial interference with cyclic imino acids at the active site. The Tyr254Ala and Tyr254Gly mutants exhibited enhanced reactivity toward cyclic imino acids by eliminating this spatial interference. Crystallographic analysis of the mutants revealed an enlarged active site, which facilitated reactions with five-membered cyclic imino acids. These mutations disrupted the electron delocalization associated with l-Tpr, thereby eliminating charge transfer and substrate inhibition. A water network was also identified near the enzyme's active site, interacting with the side chain of Tyr254. These findings provide valuable insights into substrate specificity and may facilitate the development of enzymes with broader substrate scope and enhanced catalytic activity.</p>","PeriodicalId":12187,"journal":{"name":"FEBS Open Bio","volume":" ","pages":""},"PeriodicalIF":2.3000,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"FEBS Open Bio","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1002/2211-5463.70119","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
This study investigated the reactivity of sarcosine oxidase (Sox) toward minor substrates through kinetic and structural analyses, along with mutational engineering to elucidate their reaction mechanisms. Sarcosine oxidase from Bacillus sp. (SoxB) recognizes the cyclic imino acids l-proline (l-Pro), d-proline (d-Pro), and l-thioproline (l-Tpr) as minor substrates. The reaction behavior varied depending on the substrates; notably, the absorption spectrum of l-Tpr exhibited charge transfer, which was characteristic of substrate inhibition. Crystal structures of the enzyme-substrate complexes suggested that Tyr254 causes spatial interference with cyclic imino acids at the active site. The Tyr254Ala and Tyr254Gly mutants exhibited enhanced reactivity toward cyclic imino acids by eliminating this spatial interference. Crystallographic analysis of the mutants revealed an enlarged active site, which facilitated reactions with five-membered cyclic imino acids. These mutations disrupted the electron delocalization associated with l-Tpr, thereby eliminating charge transfer and substrate inhibition. A water network was also identified near the enzyme's active site, interacting with the side chain of Tyr254. These findings provide valuable insights into substrate specificity and may facilitate the development of enzymes with broader substrate scope and enhanced catalytic activity.
期刊介绍:
FEBS Open Bio is an online-only open access journal for the rapid publication of research articles in molecular and cellular life sciences in both health and disease. The journal''s peer review process focuses on the technical soundness of papers, leaving the assessment of their impact and importance to the scientific community.
FEBS Open Bio is owned by the Federation of European Biochemical Societies (FEBS), a not-for-profit organization, and is published on behalf of FEBS by FEBS Press and Wiley. Any income from the journal will be used to support scientists through fellowships, courses, travel grants, prizes and other FEBS initiatives.