Biochemical characterization of MmgB, a gene encoding a 3-Hydroxybutyryl-CoA dehydrogenase from bacillus subtilis 168 and genetic evidence for the methylcitric acid cycle in bacillus subtilis 168

Bacillus subtilis is an industrially important organism because of its ability to produce enzymes and antibiotics on a commercial scale. It is considered the Gram positive counterpart of E. coli in terms of its genetic and molecular biological accessibility. Also, it is a model organism for the study of sporulation, an example of prokaryotic cellular differentiation. Sporulation involves several groups of genes that encode apparent metabolic pathways, many of which remain uncharacterized at the biochemical level. The mmg (mother cell metabolic gene) operon in B. subtilis strain 168 is one of these groups, and is transcribed only during an early stage of sporulation. This operon contains mmgABC, which are similar to genes from fatty acid metabolism, and mmgDE and yqiQ, which encode homologs of enzymes involved in the 2-methylcitric acid cycle. The focus of this work will be on the biochemical characterization of the mmgB protein, which is similar by sequence to 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase from a wide variety of organisms. So far, we successfully cloned and overexpressed mmgB, and purified the protein at a yield of 4.0 mg/liter of culture. A spectrophotometric assay and mass spectrometry showed that this enzyme indeed possesses 3-hydroxybutyryl-CoA dehydrogenase activity (E.C. 1.1.1.157) for the production of acetoacetyl-CoA. This oxidation specifically requires NADP+, and has an optimal pH of 9.8. We will describe these results including the steady-state kinetics that the enzyme follows. As previously said, the downstream 3 ORF's of the mmg operon - mmgD, mmgE and yqiQ are proposed to encode for the methylcitric acid cycle. Also, as a part of a master's thesis, it has been shown by the Reddick lab that mmgD encodes for a citrate/methylcitrate synthase with a substrate preference for propionyl-CoA over acetyl-CoA. This fact encouraged us even more with reference to the involvement of the mmg operon in the methylcitric acid cycle, a pathway for propionate metabolism. So, our goal was to create conditional knockout mutants of the mmg operon and study the growth characteristics of the organism by feeding studies using propionate as sole carbon source. Eventually, we would also want to perform some NMR analysis of the cultures of the mutants for the intermediates of methylcitric acid cycle/propionate metabolism