Characterization of d-threonine dehydrogenase homologues of Escherichia coli, YbbQ and YhaE

Two hypothetical proteins of Escherichia coli, YbbQ and YhaE, show high sequence similarity to d-threonine dehydrogenase. We cloned the genes encoding YbbQ and YhaE into E. coli JM109, and purified the expressed proteins to homogeneity from the E. coli clones. YbbQ consisted of two identical subunits with a molecular mass of 31 kDa, whereas YhaE was a homotetramer (native molecular mass, 124 kDa). Both enzymes required NAD+ as a coenzyme, and used serine as a substrate. d-Serine was better substrate than l-serine. YbbQ showed maximum activity at pH 11.0 for the oxidation of d-serine, whereas the optimum pH of YhaE was 10.5. These enzymes also catalyzed the oxidation of glycerate and 3-hydroxyisobutyrate. The Vmax/Km values of YbbQ for d-serine, l-serine, d-glycerate, l-glycerate, d-3-hydroxyisobutyrate, and l-3-hydroxyisobutyrate were 1.22, 0.0054, 128, 4.97, 0.0295, and 0.718 μmol min−1 mg−1 mM−1, and those of YhaE were 0.690, 0.057, 17.5, 0.650, 0.163, and 0.263 μmol min−1 mg−1 mM−1, respectively. Thus, YbbQ and YhaE are NAD+-dependent dehydrogenases acting on 3-hydroxy acids with 3-carbon chains, and d-glycerate is the best substrate for both enzymes.