Biosynthesis and Physiology of Vitamin B12 in Prokaryotes

维生素B12(钴胺)是已知的最大的非聚合天然产物之一，是一种辅助因子， 仅由某些原核生物合成的。在产生和利用B12的细菌中，有一种共生菌 固氮菌苜蓿中华根瘤菌。最近，在S体内发现了一种新的酶--blb， Meliloti，并被证明催化5，6-二甲基苯并咪唑(DMB)的生物合成 B12，其生物合成以前未知。BLOB催化黄素的裂解 在氧依赖反应中形成DMB的单核苷酸(FMN)。这项提案旨在剖析 通过稳态前动力学结合遗传分析研究这种极不寻常的酶的作用机制。 此外，这项建议试图了解平行但无关的DMB生物合成途径 被厌氧细菌感染。厌氧途径被假设为来自嘌呤生物合成的分支。 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和生化方法进行检测。该提案还旨在了解 紫花苜蓿中发生的一种新的DNA损伤反应的调节和生理意义 B12是限制性的。这一途径控制着一种改变的胞外多糖的产生。这些基因 介导这一反应将在筛查参与基因表达变化的情况下确定 胞外多糖生产。随后，将确定这一调控途径的其他靶点。 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.