Investigation and comparison of stereoisomer products of 2-methylcitrate synthase and 2-methylcitrate dehydratase in Escherichia coli strain K12 and Bacillus subtilis strain 168

Bacillus subtilis has been extensively studied and serves as a model organism for Gram-positive bacteria which can be attributed to its capacity to be genetically manipulated, form endospores, and a sequenced genome. B. subtilis undergoes a disproportionate cellular division during a process known as sporulation, which has been widely used to study more complex phenomena, such as cellular differentiation. In addition, B. subtilis is widely used by industry and a greater understanding of its metabolic pathways would be a valuable contribution to the genomic annotation in this model organism. A long-term goal of our research is to provide a more complete description of the biochemical pathways that are encoded by the mother cell metabolic gene (mmg) operon within B. subtilis. One specific objective of this research is to compare the 2-methylcitrate stereoisomer produced by 2-methylcitrate synthase, the PrpC enzyme, in Escherichia coli to the 2-methylcitrate stereoisomer produced by the homologous enzyme, MmgD, in B. subtilis. In addition, we wish to further investigate the subsequent dehydration reaction catalyzed by homologous enzymes PrpD and MmgE, which both have been found to produce (Z)-2-methylcitrate. The prpC and prpD gene products were cloned, overexpressed, and purified using a culture of E. coli strain BL21(DE3) via methods established by researchers of the Reddick Lab. The presence of the desired prpC and prpD gene products were verified by SDS-PAGE analysis and activity assays. To compare the 2-methylcitrate synthase activity of PrpC to MmgD, the reaction product was assessed by standardization with commercially available 2-methylcitrate racemate. Activity assays with PrpD and cross-species experiments were prepared in the same manner. Using this methodology, it was found that PrpC and MmgD produce either (2S, 3R)-2-methylcitrate or (3S, 2R)-2-methylcitrate. In addition, by confirming the substrates for these enzymes, we demonstrated the substrate for PrpD is not (2S, 3S)-2-methylcitrate and, therefore, PrpD does not exhibit a syn-elimination mechanism to produce (Z)-2-methylcitrate