Biosynthesis and Physiology of Vitamin B12 in Prokaryotes

描述（由申请人提供）：维生素B12是人类饮食的重要组成部分。B12缺乏症困扰着超过20%的老年人，可导致血液系统疾病、黄疸、协调性丧失和痴呆。了解这种维生素是如何合成的可能有助于全面了解和治疗B12相关疾病。与所有其他维生素不同，B12完全由微生物合成。在某些病原菌中，B12在宿主感染期间被特异性地利用。因此，研究B12在细菌中的生理学可能会导致对发病机制的见解。 直到最近，负责产生B12的“较低配体”的酶，即5，6-二甲基苯并咪唑（DMB），还是未知的。我们最近确定了BluB，一种催化共生固氮细菌苜蓿中华根瘤菌中DMB生产的酶。bluB突变体S.苜蓿在与其豆科植物宿主苜蓿建立固氮共生关系方面受到严重损害。我们的结构和生化分析表明，BluB催化的氧依赖性转化的黄素单胞苷（FMN）的DMB。拟议的工作将寻求确定这种独特转变的详细反应机制。bluB的推定同源物存在于许多细菌基因组中，表明BluB在DMB生物合成中具有普遍存在的作用。然而，一个单独的DMB生物合成途径，不使用FMN作为前体存在于厌氧细菌中，参与这一途径的酶尚未确定。厌氧DMB生物合成途径将通过遗传学，生物信息学和生物化学方法使用在研究BluB中开发的技术进行研究。此外，还观察了S.将分析苜蓿草。In S.草木犀，一种改变的胞外多糖，是响应于B12剥夺而产生的，这种响应是由DNA损伤引起的。DNA损伤诱导的改变的胞外多糖的结构，调节机制和生理相关性将被探索。总之，这些研究将有助于全面了解B12的生物合成和B12在细菌-宿主相互作用中的功能。

DESCRIPTION (provided by applicant): Vitamin B12 is a critical component of the human diet. B12 deficiency, which plagues over 20% of elderly individuals, can cause hematological disorders, jaundice, loss of coordination, and dementia. Understanding how this vitamin is synthesized may contribute to the overall knowledge and treatment of B12-related disease. Unlike all other vitamins, B12 is synthesized exclusively by microorganisms. In some pathogenic bacteria, B12 is utilized specifically during infection of the host. Therefore, studying the physiology of B12 in bacteria could lead to insights about pathogenesis. Until recently, the enzymes responsible for the production of the "lower ligand" of B12, 5,6-dimethylbenzimidazole (DMB), were unknown. We recently identified BluB, an enzyme that catalyzes the production of DMB in the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti. The bluB mutant of S. meliloti is severely impaired in establishing a nitrogen-fixing symbiosis with its legume host, alfalfa. Our structural and biochemical analyses demonstrated that BluB catalyzes the oxygen-dependent transformation of flavin mononucleotide (FMN) to DMB. The proposed work will seek to identify the detailed reaction mechanism of this unique transformation. Putative homologs of bluB exist in numerous bacterial genomes, suggesting that BluB has a ubiquitous role in DMB biosynthesis. However, a separate DMB biosynthetic pathway that does not use FMN as a precursor exists in anaerobic bacteria, and the enzymes involved in this pathway have not been identified. The anaerobic DMB biosynthetic pathway will be investigated by genetic, bioinformatic, and biochemical approaches using the techniques developed in studying BluB. Additionally, the physiological response to B12 deprivation in S. meliloti will be analyzed. In S. meliloti, an altered exopolysaccharide is produced in response to B12 deprivation, and this response is caused by DNA damage. The structure, regulatory mechanism, and physiological relevance of the DNA damage-induced altered exopolysaccharide will be explored. Together, these studies will contribute to the overall understanding of B12 biosynthesis and the function of B12 in bacteria-host interactions.