PMID: 11341914May 9, 2001Paper

Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity

Biochimica Et Biophysica Acta
S L JacquesG D Wright

Abstract

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H30...Continue Reading

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Citations

Oct 7, 2014·Letters in Applied Microbiology·N M TunM J Wu
Apr 20, 2016·Journal of Microbiology, Immunology, and Infection = Wei Mian Yu Gan Ran Za Zhi·Pei-Wen TsaiChung-Yu Lan
Jan 11, 2007·Nature Biotechnology·Alan C ChengEnoch S Huang
Jun 28, 2017·Antimicrobial Agents and Chemotherapy·Mariane C BagatinGisele de F Gauze
Jul 2, 2019·Experimental and Therapeutic Medicine·Junjun NiLijun Wu
May 7, 2015·Acta Crystallographica. Section D, Biological Crystallography·Vikas NavratnaBalasubramanian Gopal
Oct 28, 2019·Journal of Molecular Modeling·Paulo Sérgio Alves BuenoFlavio Augusto Vicente Seixas
Oct 14, 2020·Journal of Microbiology and Biotechnology·Do Hyeon KimJin Kuk Yang

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