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

Two hypothetical proteins of Escherichia coli, YbbQ and YhaE, show high seq uence similarity to D-threonine dehydrogenase. We cloned the genes encoding YbbQ and YhaE into E. coli JM109, and purified the expressed proteins to h omogeneity from the E. coli clones. YbbQ consisted of two identical subunit s with a molecular mass of 31kDa, whereas YhaE was a homotetramer (native m olecular mass, 124 kDa). Both enzymes required NAD(+) as a coenzyme, and us ed 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 V-max/K-m 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 mu mo l min(-1) mg(-1) mM(-1), and those of YhaE were 0.690, 0.057, 17.5, 0.650, 0.163, and 0.263 mu mol 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. (C) 2001 Elsevier Science B.V. All rights reserved.