The catalytically active form of histidinol dehydrogenase from <i>Salmonella typhimurium</i>

Bürger, Erich; Görisch, Helmut; Lingens, Franz

doi:10.1042/bj1810771

Cited by 16 publications

(4 citation statements)

References 15 publications

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“…The Edman degradation and ESI-MS results are also consistent with no post-translational removal of N-terminal methionine residue (131.2 Da).A value of 101.8 kDa for the molecular mass of homogeneous recombinant MtHisD was estimated by gel filtration chromatography (data not shown). This result demonstrates that MtHisD is a dimer in solution, in agreement with HisD enzymes from other organisms[22,23,44].…”

supporting

confidence: 83%

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

Nunes

Ducati

Breda

et al. 2011

Archives of Biochemistry and Biophysics

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The emergence of drug-resistant strains of Mycobacterium tuberculosis, the major causative agent of tuberculosis (TB), and the deadly HIV-TB co-infection have led to an urgent need for the development of new anti-TB drugs. The histidine biosynthetic pathway is present in bacteria, archaebacteria, lower eukaryotes and plants, but is absent in mammals. Disruption of the hisD gene has been shown to be essential for M. tuberculosis survival. Here we present cloning, expression and purification of recombinant hisD-encoded histidinol dehydrogenase (MtHisD). N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtHisD. Analytical gel filtration, metal requirement analysis, steady-state kinetics and isothermal titration calorimetry data showed that homodimeric MtHisD is a metalloprotein that follows a Bi Uni Uni Bi Ping-Pong mechanism. pH-rate profiles and a three-dimensional model of MtHisD allowed proposal of amino acid residues involved in either catalysis or substrate(s) binding.

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supporting

confidence: 83%

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

Nunes

Ducati

Breda

et al. 2011

Archives of Biochemistry and Biophysics

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“…The protein content of solutions of L-histidinol dehydrogenase was determined spectrophotometrically by using a value of 0.478 for the extinction coefficient of a 1 mg/mL solution at 280 nm (Yourno, 1968). Enzyme concentration is expressed as a molarity using the known molecular weight of the enzyme subunit, 45 823 (Kohno & Gray, 1981;Burger et al, 1979). Where noted, concentration is also expressed as molarity of the dimer.…”

Section: Methodsmentioning

confidence: 99%

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

Grubmeyer¹,

Gray²

1986

Biochemistry

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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.

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“…Protein determination used the known extinction coefficient e^sonm °f 0.478 (Yourno, 1968). The subunit molecular weight of 45 823 from protein sequencing (Kohno & Gray, 1981) was used to calculate subunit molarity in solutions of the stable dimer (Loper, 1968;Burger et al, 1979). The standard continuous assay of enzyme activity was performed as described (Grubmeyer & Gray, 1986) and employed 1-mL mixtures containing 50 Mmol of glycine-NaOH, 2 Mmol of histidinol, 0.5 Mmol of MnCl2, and 10 Mmol of NAD, brought to pH 9.2 with NaOH.…”

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confidence: 99%

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Grubmeyer¹,

Chu²,

Insinga³

1987

Biochemistry

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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)

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The catalytically active form of histidinol dehydrogenase from Salmonella typhimurium

Cited by 16 publications

References 15 publications

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

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

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

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