Purification and in vitro complementation of mutant histidinol dehydrogenases

Lee, S Y; Grubmeyer, Charles

doi:10.1128/jb.169.9.3938-3944.1987

Cited by 15 publications

(10 citation statements)

References 25 publications

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“…Finally, it has long been known that mutations in the hisD gene fall into two major groups that complement in vivo to produce active enzyme (Greeb et al, 1971), suggesting that the mutations might characterize regions involved in the two HDH half-reactions. However, we have recently used in vitro complementation of purified mutant HDH to show that neither mutant class catalyzed the NAD/NADH exchange reaction reflective of the first oxidation (Lee & Grubmeyer, 1987).…”

Section: Discussionmentioning

confidence: 99%

Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping

Grubmeyer¹,

Insinga²,

Bhatia³

et al. 1989

Biochemistry

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The stereochemistry of the L-histidinol dehydrogenase reaction was determined to be R at NAD for both steps, confirming previous results with a fungal extract [Davies, D., Teixeira, A., & Kenworthy, P. (1972) Biochem. J. 127, 335-343]. NMR analysis of monodeuteriohistidinols produced by histidinol/NADH exchange reactions arising via reversal of the alcohol oxidation reaction indicated a single stereochemistry at histidinol for that step. Comparison of vicinal coupling values of the exchange products with those of L-alaninol and a series of (S)-2-amino-1-alcohols allowed identification of the absolute stereochemistry of monodeuteriohistidinols and showed that histidinol dehydrogenase removes first the pro-S then the pro-R hydrogens of substrate histidinol. The enzyme stereochemistry was confirmed by isotope effects for monodeuteriohistidinols as substrates for the pro-R-specific dehydrogenation catalyzed by liver alcohol dehydrogenase. Active site mapping was undertaken to investigate substrate-protein interactions elsewhere in the histidinol binding site. Critical binding regions are the side-chain amino group and the imidazole ring, whose methylation at the 1- or 2-position caused severe decreases in binding affinity. Use of alternative substrates further clarified active site interactions with the substrate. Compounds in which the alpha-amino group was replaced by chloro, bromo, or hydrogen substituents were not substrates of the overall reaction at 1/10,000 the normal rate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping

Grubmeyer¹,

Insinga²,

Bhatia³

et al. 1989

Biochemistry

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“…In the case of alkaline phosphatase, Fan et al (1966) found nonrandom association of various mutant forms, and specific activities for the complemented products varied. In histidinol dehydrogenase (Lee & Grubmeyer, 1987) heterodimers were strongly favored in mixtures of complementing subunits because the ability of the complemented heterodimer to bind Mn2+ allowed for a thermodynamic pull toward heterodimer formation.…”

Section: Biochemistrymentioning

confidence: 99%

Structure and Function of Salmonella typhimurium Orotate Phosphoribosyltransferase: Protein Complementation Reveals Shared Active Sites

Ozturk

Dorfman²,

Scapin³

et al. 1995

Biochemistry

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A solvent-exposed loop, comprising residues 98-119 of S. typhimurium orotate phosphoribosyltransferase (OPRTase), is at the subunit interface of the dimeric enzyme, and its amino acid side chains potentially contact active sites on either subunit. A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme.OMP [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] and enzyme.PRPP.orotate complexes [Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754]. Lys-103, which is essential for activity [Ozturk, D. H., Dorfman, R. H. Scapin, G., Sacchettini, J. C., & Grubmeyer, C. (1995) Biochemistry 34, 10755-10763], may thus be functional in the active site formed by the adjacent subunit. Asp-125 is an essential residue that is in the middle of the active site. Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produced approximately 21-23% of the enzymatic activity of the wild-type OPRTase. Heterodimer formation in the complemented mixtures was evidenced by various physical methods. Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit. As predicted from the three-dimensional structure, increased activity resulting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtures of D125N and either K73A or K73Q mutants. Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme. A K103A/D125N double mutant enzyme was also constructed and was able to inactivate wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…1A). Previously described HisD enzymes are homodimers [11,12] containing one Zn 2+ per subunit [11]; and they are 0003-9861/$ -see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2011.05.020 therefore examples of metalloenzymes.…”

Section: Introductionmentioning

confidence: 99%

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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Purification and in vitro complementation of mutant histidinol dehydrogenases

Cited by 15 publications

References 25 publications

Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping

Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping

Structure and Function of Salmonella typhimurium Orotate Phosphoribosyltransferase: Protein Complementation Reveals Shared Active Sites

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

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