Biosynthesis and Physiology of Vitamin B12 in Prokaryotes

维生素B12（钴胺素）是已知最大的非聚合天然产物之一，并且是维生素B12的辅因子。 只有某些原核生物才能合成在产生和利用B12的细菌中， 固氮菌苜蓿中华根瘤菌。最近，一种新的酶，BluB，被发现在S， 草木樨并显示催化5，6-二甲基苯并咪唑（DMB）的生物合成，DMB是草木樨的下轴配体。 B12，其生物合成以前未知。BluB催化黄素的断裂 在氧依赖性反应中，可使用单甘肽（FMN）来形成DMB。这项建议旨在剖析 通过预稳态动力学结合遗传分析，研究了这种极不寻常的酶的作用机制。 此外，该提案旨在了解所利用的平行但不相关的DMB生物合成途径 由厌氧细菌。厌氧途径被假设为嘌呤生物合成途径的分支。 而不是使用FMN作为前体。参与这一途径的候选基因将被确定 通过生物信息学和遗传学和生物化学方法测试。该提案还旨在了解 调节和生理意义的一种新的DNA损伤反应在S。草木犀，当 B12是有限的。该途径控制改变的胞外多糖的产生。的基因 介导这种反应的基因将在筛选中被鉴定出来，以改变参与这种反应的基因表达。 胞外多糖的产生。随后，将确定这一调控途径的其他靶点。 总之，这些实验将有助于理解B12的生物合成和功能， 细菌，并可能导致人类营养和疾病治疗的进步。

Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products and is a cofactor that is synthesized only by certain prokaryotes. Among the bacteria that produce and utilize B12 is the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Recently, a novel enzyme, BluB, was discovered in S, meliloti and shown to catalyze the biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of B12 whose biosynthesis was previously unknown. BluB catalyzes the fragmentation of flavin mononucleotide (FMN) to form DMB in an oxygen-dependent reaction. This proposal aims to dissect the mechanism of this highly unusual enzyme by pre-steady state kinetics combined with genetic analyses. Additionally, this proposal seeks to understand the parallel but unrelated DMB biosynthetic pathway utilized by anaerobic bacteria. The anaerobic pathway is hypothesized to branch from the purine biosynthetic pathway rather than using FMN as a precursor. Candidate genes involved in this pathway will be identified by bioinfonnatics and tested by genetic and biochemical methods. The proposal also aims to understand the regulation and physiological significance of a novel DNA damage response in S. meliloti that occurs when B12 is limiting. This pathway controls the production of an altered exopolysaccharide. The genes that mediate this response will be identified in a screen for altered expression of genes involved in exopolysaccharide production. Subsequently, other targets of this regulatory pathway will be identified. Together these experiments will contribute to the understanding of the biosynthesis and function of B12 in bacteria and may lead to advances in human nutrition and disease treatment.