Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
J Bacteriol. 2018 May 24;200(12). doi: 10.1128/JB.00022-18. Print 2018 Jun 15.
The actinobacterium splits riboflavin (vitamin B) into lumichrome and d-ribose. However, such degradation by other bacteria and the involvement of a two-component flavin-dependent monooxygenase (FMO) in the reaction remain unknown. Here we investigated the mechanism of riboflavin degradation by the riboflavin-assimilating alphaproteobacterium (formerly ). We found that adding riboflavin to bacterial cultures induced riboflavin-degrading activity and a protein of the FMO family that had 67% amino acid identity with the predicted riboflavin hydrolase (RcaE) of MF109. The genome clustered genes encoding the predicted FMO, flavin reductase (FR), ribokinase, and flavokinase, and riboflavin induced their expression. This finding suggests that these genes constitute a mechanism for utilizing riboflavin as a carbon source. Recombinant FMO (rFMO) protein of oxidized riboflavin in the presence of reduced flavin mononucleotide (FMN) provided by recombinant FR (rFR), oxidized FMN and NADH, and produced stoichiometric amounts of lumichrome and d-ribose. Further investigation of the enzymatic properties of rFMO indicated that rFMO-rFR coupling accompanied O consumption and the generation of enzyme-bound hydroperoxy-FMN, which are characteristic of two-component FMOs. These results suggest that FMO is involved in hydroperoxy-FMN-dependent mechanisms to oxygenize riboflavin and a riboflavin monooxygenase is necessary for the initial step of riboflavin degradation. Whether bacteria utilize either a monooxygenase or a hydrolase for riboflavin degradation has remained obscure. The present study found that a novel riboflavin monooxygenase, not riboflavin hydrolase, facilitated this process in The riboflavin monooxygenase gene was clustered with flavin reductase, flavokinase, and ribokinase genes, and riboflavin induced their expression and riboflavin-degrading activity. The gene cluster is uniquely distributed in species and actinobacteria, which have exploited an environmental niche by developing adaptive mechanisms for riboflavin utilization.
放线菌将核黄素（维生素 B）分解为光色素和 D-核糖。然而，其他细菌的这种降解以及反应中涉及的双组分黄素依赖性单加氧酶（FMO）仍然未知。在这里，我们研究了参与黄素同化的 α 变形菌（以前称为）分解核黄素的机制。我们发现，向细菌培养物中添加核黄素会诱导核黄素降解活性和一种与 MF109 中预测的核黄素水解酶（RcaE）具有 67%氨基酸同一性的 FMO 家族蛋白。基因组聚类编码预测的 FMO、黄素还原酶（FR）、核酮激酶和黄素激酶的基因，并诱导它们的表达。这一发现表明这些基因构成了一种利用核黄素作为碳源的机制。来自 的重组 FMO（rFMO）蛋白在重组 FR（rFR）提供的还原黄素单核苷酸（FMN）的存在下氧化核黄素，产生等摩尔量的光色素和 D-核糖。进一步研究 rFMO 的酶学特性表明，rFMO-rFR 偶联伴随着 O 消耗和酶结合的过氧黄素单核苷酸的产生，这是双组分 FMO 的特征。这些结果表明，FMO 参与依赖过氧黄素单核苷酸的机制来氧化核黄素，并且核黄素单加氧酶是核黄素降解初始步骤所必需的。细菌是否利用单加氧酶或水解酶来降解核黄素仍然不清楚。本研究发现，一种新型核黄素单加氧酶，而不是核黄素水解酶，在 中促进了这一过程。核黄素单加氧酶基因与黄素还原酶、黄素激酶和核酮激酶基因聚类，并且核黄素诱导它们的表达和核黄素降解活性。该基因簇在 物种和放线菌中具有独特的分布，它们通过开发用于核黄素利用的适应性机制来利用环境小生境。
