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

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of t hreonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overpro duced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate s emialdehyde to homoserine (Hse) using either NADH or NADPH with k(cat)/K-m in the order of 10(6-7) M-1 s(-1) at pH 7.5. The rate constant of the rever se direction (Hse oxidation) was also significant at pH 9.0 (k(cat)/K-m app roximate to 10(4-5) M-1 s(-1)) but was minimal at pH 7.5. Chemical modifica tion of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activit y that could be obviated by the presence of substrates. UV difference spect ra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consi stent with the modification of two histidines (I-Iis) per subunit. Amino ac id sequence alignment of HSD illustrated the conservation of two His residu es among HSDs. These residues, His79 and His309, were substituted to alanin e (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while K-cat/K-m for HSD H309A decreased by almost tw o orders of magnitude. The recent determination of the X-ray structure of H SD revealed that His309 is located at the dimer interface [B. DeLaBarre, P. R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-24 4]. The His309Ala mutant enzyme was found in very high molecular weight com plexes rather than the expected dimer by analytical gel filtration chromato graphy analysis. Thus the invariant His309 plays a structural rather than c atalytic role in these enzymes. (C) 2001 Elsevier Science B.V. All rights r eserved.