The CbiB Protein of Salmonella enterica is an Integral Membrane Protein Involved in the Last Step of the De Novo Corrin Ring Biosynthetic Pathway

Department of Bacteriology, University of Wisconsin-Madison

^* To whom correspondence should be addressed. Email: escalante{at}bact.wisc.edu.

	Abstract

We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate (AdoCbi-P), the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B₁₂) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted CbiB to 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 for 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 to 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.