Purification and in vitro complementation of mutant histidinol dehydrogenases.
The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the hisO1242 (constitutive overproducer) attenuator mutation and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl2, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Neither mutant protein showed negative complementation with wild-type enzyme. The Vmax for hybrid histidinol dehydrogenase was 11% of that for native enzyme, with only minor changes in Km values for substrate or coenzyme. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound 54Mn2+ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound 54Mn2+ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 2-mercaptoethanol was prevented by low levels of MnCl2. It thus appears that mutant histidinol dehydrogenase molecules bind metal ion poorly. The complementation procedure may allow for formation of a functional Mn2+-binding site, perhaps at the subunit interface.
Lee
S Y
SY
Grubmeyer
C T
CT
D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D009154:Mutation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics
D008345:Manganese; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase