Department of Plant & Microbial Biology, University of California, Berkeley, California 94720-3102, USA.
Protein Sci. 2012 Jun;21(6):839-49. doi: 10.1002/pro.2068. Epub 2012 Apr 23.
The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.
“黄素破坏酶”BluB 催化前所未有的黄素单核苷酸 (FMN) 转化为 5,6-二甲基苯并咪唑 (DMB)，DMB 是维生素 B(12) 的组成部分。由于其不寻常的化学性质，这种转化的机制一直难以捉摸。本研究报告了 12 种突变形式的 BluB 的鉴定，这些突变形式的催化功能严重降低，但大多数仍保留结合黄素的能力。“黄素破坏酶”BluB 是一种不寻常的酶，它在氧气存在下破坏黄素辅因子 FMNH(2)，产生 5,6-二甲基苯并咪唑 (DMB)，DMB 是维生素 B(12)（钴胺素）的下轴向配体。尽管 BluB 在序列和结构上与硝基还原酶和黄素氧化还原酶家族相似，但 BluB 是已知唯一能够破坏黄素异咯嗪环的酶。为了探索参与这种不寻常反应的催化残基，在细菌 Sinorhizobium meliloti 的遗传筛选中鉴定出了在 DMB 生物合成中受损的 BluB 突变体。在筛选中鉴定出的 16 个独特点突变中，大多数位于活性位点的保守残基或提议屏蔽活性位点与溶剂的独特“盖”域中。12 种纯化突变蛋白的稳态酶测定显示，所有突变体的 DMB 合成均显著减少，其中 8 种完全缺乏 DMB 产生。其中 10 种突变体对氧化和还原 FMN 的结合亲和力较弱，尽管只有两种对复合物稳定性有显著影响。这些结果表明 BluB 具有独特的 FMNH(2) 片段化能力，并证明了 BluB 活性位点对结构扰动的敏感性。这项工作为该酶的机制研究奠定了基础，并进一步加深了我们对 BluB 的结构-功能关系的理解。
