Journal
JOURNAL OF BACTERIOLOGY
Volume 189, Issue 21, Pages 7697-7708Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.01090-07
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Funding
- NIGMS NIH HHS [GM40313, R01 GM040313, F31 GM064009, F31-GM64009, R37 GM040313] Funding Source: Medline
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We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B-12) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that Chill would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but L-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.
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