Site-Directed Mutagenesis of Histidine-90 inEscherichia colil-Threonine Dehydrogenase Alters Its Substrate Specificity

The First Crystal Structure of l-Threonine Dehydrogenase

“…32,33 Without the significant hydrogen bonds, the high affinity of PhTDH to NAD(H) was observed. These results strongly support the proposed ordered Bi Bi mechanism for TDH, 3,31 i.e. NAD + combines with the enzyme prior to the binding of threonine, and 2-amino-3-oxobutyrate leaves from the enzyme before the release of NADH.…”

“…In the case of EcTDH, it has been reported that Glu88 and His90 are important for the substrate specificity. 30,31 In particular, His90 was reported not to coordinate the catalytic zinc ion but to modulate the substrate specificity of EcTDH. 31 In the case of PhTDH, Glu92 (Glu88 in EcTDH) forms an ionic bond with Arg294 and is not located in the cleft.…”

“…30,31 In particular, His90 was reported not to coordinate the catalytic zinc ion but to modulate the substrate specificity of EcTDH. 31 In the case of PhTDH, Glu92 (Glu88 in EcTDH) forms an ionic bond with Arg294 and is not located in the cleft. Glu92, His94 (His90 in EcTDH), Glu152 and Arg294 forms hydrogen or ionic bonds near the electron transfer site of NAD(H) and seem to be related to the substrate specificity.…”

“…This position corresponding to SO 4 2− seems to be important for the substratebinding site (carboxyl residue and amino residue in threonine), as observed in the case of EcTDH. 31 Most of the ADH structures have been solved as the apo-form. The finding that PhTDH was crystallized in the absence of NAD(H) indicates that NAD (H) in the recombinant enzyme is derived from E. coli cells and the binding affinity of PhTDH to NAD(H) is high, as suggested by the kinetical data in the previous paper.…”

A Vibrio cholerae autoinducer–receptor pair that controls biofilm formation

“…1). Previous Tdh studies in Escherichia coli revealed two critical amino acid residues required for catalytic activity: Cys-38 and His-90 33 . To test if Tdh catalytic activity is necessary for production of the molecule required for VqmA-directed activation of PvqmR::mkate2 expression, we mutated the corresponding residues in the V. cholerae Tdh enzyme to alanine (C40A) and arginine (H92R), respectively.…”