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（钴胺素）是已知最大的非聚合天然产品之一，是一种辅助因子， 仅由某些原核生物合成。产生和利用 B12 的细菌中有共生菌 固氮细菌苜蓿中华根瘤菌。最近，在S中发现了一种新的酶BluB， 苜蓿草并显示可催化 5,6-二甲基苯并咪唑 (DMB)（DMB 的下轴配体）的生物合成 B12 的生物合成以前未知。 BluB 催化黄素裂解 单核苷酸（FMN）在氧依赖性反应中形成 DMB。该提案旨在剖析 通过前稳态动力学与遗传分析相结合，研究了这种极不寻常的酶的机制。 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 通过厌氧细菌。推测厌氧途径是从嘌呤生物合成分支出来的 途径而不是使用 FMN 作为前体。将鉴定参与该途径的候选基因 通过生物信息学并通过遗传和生化方法进行测试。该提案还旨在了解 苜蓿中华根瘤菌中一种新型 DNA 损伤反应的调节和生理意义 B12 是有限的。该途径控制改变的胞外多糖的产生。这些基因 介导这种反应的基因将在筛选中被识别，以改变相关基因的表达 胞外多糖的产生。随后，将确定该监管途径的其他目标。 这些实验共同将有助于了解 B12 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。