Alternation in Substrate Specificity of Amino Acid Dehydrogenases based on the Structural Analyses

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According to the structural analysis of glutamate dehydrogenase (GluDH) of Clostridium symbiosum, we studied the alternation of substrate specificity of phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus to leucine dehydrogenase (LeuDH). We focused on residues which are not common in the amino acid dehydrogenases around the pocket for the substrate. 163A and 377V which are found in GluDH and LeuDH were changed to G and L in PheDH, respectively. The modified GluDH showed lower activity toward L-Phe, where as the activity toward aliphatic amino acids were increased.Opine dehydrogenase (ODH) from a soil isolate Arthrobacter sp. strain 1C showed similarity toward 40-kDa Protein, D-lysopine dehydrogenase, D-nopaline dehydrogenase found in plant crown gall. ODH was highly purified from E. coli transformant. Crystals of ODH, obtained in the presence or absence of co-factor and substrate, have been shown to diffract to beyond 1.8Å resolution. We carried out site-directed mutagenesis on these residues and other ones in both domains, and the results indicated that substitution of either of these six residues or Arg-143, Lys-156, His-222, or Tyr-280 impaired catalysis significantly. Steady-state kinetic interaction and catalysis. An opine analog N-[1-(S)-(carboxyl)ethyl]-(S)-phenylalanine competitively inhibited the wild type and most mutants with the exception of Arg143 mutants, Further, we observed a sulfate ion bound in the vicinity of His-202, Tyr-222, Arg-292 and Tyr-293 in the putative active site. We modeled a pyruvate in the sulfate binding site and a catalytic mechanism of ODH to resemble that of the 2-hydroxy acid dehydrogenases is proposed.