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.

Public Health Relevance

Vitamin B12 (cobalamin) is a critical component ofthe human diet. B12 deficiency plagues a significant proportion of the human population including over 20% of elderly individuals. B12 is the only vitamin that is produced exclusively by prokaryotes. Understanding the biosynthesis ahd physiological function of B12 in bacteria is essential to human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Transition Award (R00)
Project #
5R00GM083343-05
Application #
8006412
Study Section
Special Emphasis Panel (NSS)
Program Officer
Anderson, Vernon
Project Start
2007-12-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2012-12-31
Support Year
5
Fiscal Year
2011
Total Cost
$244,045
Indirect Cost
Name
University of California Berkeley
Department
Other Basic Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Degnan, Patrick H; Barry, Natasha A; Mok, Kenny C et al. (2014) Human gut microbes use multiple transporters to distinguish vitamin B₁₂ analogs and compete in the gut. Cell Host Microbe 15:47-57