Enzyme mediated 2-methyl-3-oxosuccinate tautomerization is required for branched-chain amino acid synthesis
A substrate does not need to have a precise shape to fit the active site of the enzyme in many cases (1). Enzymes will not only act on their canonical substrates, but also compounds that are structurally similar. When this “promiscuous” activity occurs, substrates can be erroneously metabolized to damaged products with toxic effects on the cell (2). Metabolite repair enzymes function to convert these damaged metabolites to neutral or beneficial compounds in order to maintain appropriate metabolic pools and fluxes (3). We have previously determined that the FAH-domain containing enzyme oxaloacetate tautomerase (OAT1) will convert the damage product enol-oxaloacetate (e-OAA) to the TCA cycle intermediate keto-oxaloacetate (k-OAA). Through a comparative genomic analysis of several different bacterial genomes, we found the OAT1 clustered very strongly with the branched chain amino acid (BCAA) synthesis gene 3-isopopylmalate dehydrogenase. We hypothesized that OAT1 would increase the rate of tautomerization of the BCAA synthesis intermediate 2-methyl-3-oxosuccinate, which is produced by 3-isopropylmalate dehydrogenase and formerly thought to spontaneously undergo tautomerization and decarboxylation. To test this, we purified 3-isopropylmalate dehydrogenase (E.coli LeuB) and used spectrometric assays to measure the reaction with and without OAT1. The reaction was also analyzed by high-performance liquid chromatography (HPLC). Through these methods, we found that OAT1 facilitates the tautomerization of 2-methyl-3-oxosuccinate, adding to the comparative genomic evidence that it plays a role in BCAA synthesis.