Site-directed mutagenesis shows that the conserved cysteine residues of histidinol dehydrogenase are not essential for catalysis.
Histidinol dehydrogenase (HDH) catalyzes two sequential oxidation reactions to produce histidine from histidinol via histidinaldehyde. In HDH proteins so far reported, two Cys residues are conserved. From the results of the studies on Salmonella typhimurium HDH, it has been proposed that one of these two conserved Cys residues is involved in the thiohemiacetal formation at the aldehyde oxidation step [Grubmeyer and Gray (1986) Biochemistry 25, 4778-4784]. To clarify the reaction mechanism, we investigated the role of the conserved Cys residues by site-directed mutagenesis in cabbage HDH. Thus, Cys-112, that corresponds to the catalytic Cys residue of the Salmonella enzyme, and the other conserved one, Cys-149, were replaced with Ala, Ser, or Phe. All the Cys-112 mutant HDHs catalyzed both the alcohol dehydrogenase and aldehyde dehydrogenase reactions, producing 1 mol of L-histidine during the reduction of 2 mol of NAD+, as did the wild type HDH. Site-directed mutagenesis at Cys-149 did not cause significant changes in the catalytic properties, either. These observations, together with the results of detailed comparison of the catalytic properties of mutant HDHs, clearly indicate that neither Cys-112 nor Cys-149 is involved in the reaction, and ruled out the involvement of thiohemiacetal formation in the histidinol dehydrogenase reaction.
Nagai
A
A
International Research Laboratories, Ciba-Geigy Japan, Ltd., Hyogo.
Kheirolomoom
A
A
Ohta
D
D
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000494:pharmacology; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D017343:Genes, Plant; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000494:pharmacology
C060274:4-(N-(acetoxyethyl)-N-methylamino)-7-nitro-2,1,3-benzoxadiazole ubiquinone; D014451:Ubiquinone; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D009327:4-Chloro-7-nitrobenzofurazan; D003545:Cysteine