Biosynthesis and Physiology of Vitamin B12 in Prokaryotes

原核生物维生素 B12 的生物合成和生理学

• 批准号: 8006412
• 负责人: Michiko E. Taga
• 金额: $ 24.4万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8006412
• 关键词: Anabolism; Anaerobic Bacteria; Bacteria; Biochemical; Biological Factors; Candidate Disease Gene; Catalysis; Cellular Stress; Cleaved cell; Cobalamin; DNA Damage; Defect; Diet; Disease; Elderly; Enzymes; Eubacterium; Flavin Mononucleotide; Flavins; Gene Expression; Genes; Genetic Screening; Genetic screening method; Genome; Health; Human; Individual; Kinetics; Label; Lead; Ligands; Mass Spectrum Analysis; Mediating; Melilotus; Methods; Nitrogen; Oxygen; Pathway interactions; Phenotype; Physiological; Physiology; Plague; Population; Process; Production; Prokaryotic Cells; Purines; Reaction; Regulation; Regulatory Pathway; SOS Response; Scheme; Sinorhizobium meliloti; Son of Sevenless Proteins; Spectrophotometry; Structure; Time; Vitamin B 12; Vitamins; base; cofactor; genetic analysis; isoalloxazine; mutant; novel; nutrition; purine; research study; response

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.

维生素B12（钴胺）是最大的已知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。在产生和利用B12的细菌中是共生 固氮细菌sinorhizobium meliloti。最近，在S中发现了一种新型酶Blub， Meliloti并显示出催化5,6-二甲基苯甲酰唑（DMB）的生物合成，较低的轴向配体 B12的生物合成以前未知。 Blub催化黄素的碎片化 单核苷酸（FMN）以氧依赖性反应形成DMB。该建议旨在剖析 这种高度不寻常的酶的机制由稳态的状态动力学与遗传分析相结合。 此外，该提案旨在理解所利用的平行但无关的DMB生物合成途径 通过厌氧细菌。假设厌氧途径是从嘌呤生物合成的分支 途径而不是使用FMN作为前体。将确定参与此途径的候选基因 通过生物促成，并通过遗传和生化方法进行了测试。该提案还旨在了解 新型DNA损伤反应在Meliloti中发生的调节和生理意义 B12是有限的。该途径控制着改变的外多糖的产生。那个基因 调解此反应将在屏幕上确定，以改变涉及的基因表达 外多糖生产。随后，将确定此法规途径的其他目标。 这些实验将加在一起有助于理解B12在B12中的生物合成和功能 细菌，可能会导致人类营养和疾病治疗的进步。