Cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Abstract

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.

Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

“…The enzyme histidinol dehydrogenase also catalyzes the NAD ϩ -dependent 2-fold oxidation of an alcohol to an acid (without the release of aldehyde) and was generally thought to use a mechanism similar to that of UDP-glucose dehydrogenase (1). Treatment of the enzyme with 7-chloro-4-nitro-2,1,3-benzoxadiazole resulted in the covalent labeling of an active site cysteine and the loss of enzyme activity (32). This seemed to indicate that Cys-116 supplied a thiol that participated in the second oxidation step.…”

Purification and in vitro complementation of mutant histidinol dehydrogenases

“…Under the high-density conditions used to grow hisO1242 hisD+ cells (11), both mutant cell types failed to show a band corresponding to HDH on SDS-polyacrylamide gels and extracts failed to complement. However, both protein and complementation activity were found in normal-density overnight cultures, which were therefore used for all mutant protein preparations.…”

“…For enzyme preparation, 12 500-ml cultures of bacteria were grown in 2-liter shaker flasks at 37°C in LB broth (6). Growth of mutant strains in the high-density system described earlier (11) Isolation of mutant HDH. The procedure followed to isolate the mutant enzymes was a modified version of that used by Yourno and Ino (25).…”

Zinc metalloenzymes as new targets against the bacterial pathogen Brucella

“…During the analysis of the B. suis virulome, our group identified L-HDH as a virulence gene essential for the intramacrophagic replication [13]. Although L-HDH represents an interesting target for the development of new antibacterials, to date, HDH has been cloned and characterized from only three bacteria, E. coli [43], S. typhimurium [35,44,45] and, more recently, B. suis within our research groups [46]. The possibility to selectively inhibit the bacterial enzyme L-HDH has opened a new route toward the discovery of new antibacterials.…”