Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady-state investigations of the mechanism of histidinol dehydrogenase.

Initial velocities of the histidinol dehydrogenase reaction (EC 1.1.1.23) were measured as a function of the concentrations of the substrates histidinol and NAD+ and in the presence and absence of the product NADH. The data are consistent with a Bi Uni Uni Bi Ping Pong mechanism. The kinetic constants of this mechanism were determined; Km for histidinol was found to be 14 microM and for NAD+ 0.7 mV; Ki for NAD+ was 0.4 mM.     

Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.

The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.     

Binding of histidinal to histidinol dehydrogenase

One molecule of the enzymatic intermediate histidinal is firmly bound per subunit of histidinol dehydrogenase (EC 1.1.1.23) and protected against decomposition. The dissociation rate constant of the histidinal--histidinol dehydrogenase complex is estimated as 2.5 X 10(-5) S-1. Steady-state kinetic measurements studying the oxidation of histidinal to histidine and the reduction of histidinal to histidinol allow to calculate the association rate constants for histidinal. For both reactions the association rate constant is found as 1.9 X 10(6) M-1 S-1. Thus the dissociation constant of the histidinal--histidinol dehydrogenase complex is estimated to be of the order of 1.4 X 10(-11) M.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase

A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. [¹⁴C]histidine, from the substrate, [¹⁴C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.[¹⁴C]histidinol is obtained in high yields through conversion of uniformly labeled ¹⁴C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated [¹⁴C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system.     

The catalytically active form of histidinol dehydrogenase from Salmonella typhimurium.

The active-enzyme-sedimentation procedure was used to identify the catalytically competent form of histidinol dehydrogenase (EC 1.1.1.23) isolated from Salmonella typhimurium. At pH 9.4 the active species has a sedimentation coefficient S20,W of 5.4S, indicating that the dimer with a mol.wt. of approx. 83 000 is the enzymically active form.     

Aldehyde dismutase activity of yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (EC 1.1.1.1) is able to catalyze the oxidation of acetaldehyde by NAD+ with a concomitant formation of ethanol, at pH 8.8 and pH 7.1; the stoichiometry of aldehyde oxidation vs. ethanol formation is 2:1. This enzymatic reaction obeys the Michaelis-Menten kinetics and was characterized by a high KM for acetaldehyde (68 mM) and a low kcat (2.3 s–1), at pH 8.8, 22°C. There is no visible burst of NADH during the reaction, from pH 7.1–10.1. Therefore, we have concluded that the enzyme catalyzes an apparent dismutation of two molecules of acetaldehyde into a molecule of acetic acid and a molecule of ethanol.     

Overexpression of plant histidinol dehydrogenase using a baculovirus expression vector system.

A cDNA encoding cabbage histidinol dehydrogenase, including the chloroplast transit peptide sequence, was overexpressed using a baculovirus expression vector system. The maximum level of the expression of histidinol dehydrogenase was reached 5 days after infection of the insect cells. Two forms of recombinant histidinol dehydrogenase with molecular masses of 53 and 52 kDa, respectively, were obtained by a one-step purification from the cell homogenate. Compared with the 52-kDa form, the 53-kDa form contained 10 additional amino acids at the N-terminus derived from the transit peptide. By incubating the cell homogenate for 2 h at 30 degrees C, the 53-kDa form could be completely converted to the 52-kDa form. This conversion was blocked by leupeptin. Eighty percent of the converted 52-kDa form had Cys at position 31 at the N-terminal amino acid and the rest had Met 33. Kinetic properties of the recombinant enzyme were virtually identical to those of histidinol dehydrogenase isolated from cabbage plants. The overexpression of recombinant cabbage histidinol dehydrogenase in insect cells, the proteolytic processing of the preprotein next to the N-terminus (compared to the mature cabbage enzyme), and its easy purification allow the preparation of large amounts of the active enzyme for structural and functional studies.     

Mechanisms of action of histidinol dehydrogenase and UDP-Glc dehydrogenase. Evidence that the half-reactions proceed on separate subunits.

Histidinol dehydrogenase and UDP-Glc dehydrogenase catalyze 4-electron dehydrogenations that convert primary alcohol groups to the corresponding acids. Both reactions proceed in two distinct steps involving the oxidation of the primary alcohol to a bound form of the intermediate aldehyde, followed by oxidation of this to the corresponding acid. The enzymes have subunit structure, the former is made up of two subunits and the latter of six (beef liver enzyme). Evidence is presented that the two half-reactions proceed independently of the overall reaction. Histidinol dehydrogenase preparations that approach total dissociation into subunits also approach total inhibition of the overall reaction, while the second half reaction is completely unaffected and 50% of the first half-reaction survives. Further, the fraction of overall activity surviving in partially dissociated preparations follows the weight fraction of residual dimer. UDP-Glc dehydrogenase behaves in an analogous fashion. These data are interpreted on the basis that both enzymes function by carrying out first oxidation step at a site on one subunit and then pass the intermediate to a vicinal site on the adjacent subunit, where the reaction is completed.     

Structure based design of novel inhibitors for histidinol dehydrogenase from Geotrichum candidum

Histidinol dehydrogenase, the product of the HisD gene, mediates the final step in the histidine biosynthetic pathway. This enzyme has captured attention for drug discovery studies in past few years. Recently, our group cloned and expressed Geotrichum candidum histidinol dehydrogenase and successful screening of substrate analog inhibitors of histidinol dehydrogenase led to some antifungal compounds with IC₅₀ values in micromolar range. In this study, we have done docking analysis of these antifungal agents in G. candidum. Two new compounds were designed based on the docking results and these compounds turned out to be potent inhibitors of G. candidum histidinol dehydrogenase, showing IC₅₀ values as low as 3.17 μM.     

A specific peroxidase-coupled activity stain for diamine oxidases.

A specific peroxidase-coupled activity staining method for diamine oxidase (DAO) was developed. Diaminobenzidine was found to inhibit DAO and to give rise to unspecific staining. Among several other reagents 4-Cl-1-naphthol was found to be most suitable. Using specific activity staining DAO could be visualized in polyacrylamide gels as a high-molecular-weight complex, which could be dissociated by Tween 20 but not by NP-40, Triton X-100, or Chaps.     

The purification and properties of histidinol dehydrogenase from Neurospora crassa

1. A procedure is described for the purification of l-histidinol dehydrogenase (l-histidinol-NAD oxidoreductase, EC 1.1.1.23) from Neurospora crassa. 2. The enzyme, as purified, has a sedimentation coefficient, S(20), of 7.1s and a molecular weight of 81 000. Considerable variation is possible in the state of polymerization of the enzyme, giving rise to observed molecular weights from 40 000 to 240 000. 3. Several kinetic parameters of the enzyme have been determined. The enzyme is maximally active at pH9.8; the K(m) (NAD) is 13.0x10(-5)m and K(m) (histidinol) is 8.2x10(-6)m. The enzyme is highly specific, does not oxidize a range of amino alcohols and other aliphatic alcohols nor reduce NADP and has no demonstrable affinity for histidine. The turnover number is 49 moles of NAD reduced/min./mole of enzyme (mol.wt. 40 000).