413 Acta Crvst. (1998). D54, 413--415 Crystallization and preliminary X-ray diffraction studies of homoserine dehydrogenase from Saccharomyces cerevisiae BYRON DELABARRE, SUZANNE L. JACQUES,CNI'HARINE E. PRATT, t DEREKA. Rurn, GERARDD. WRIGHT AND ALBERTM. BERGHUIS* at Department of Biochemistry, McMaster University, 1200 Main St. West, ttamilton, Ontario, Canada L8N 3Z5. E-mail: l~erghuis@rncrnaster.ca (Received 23 July 1997: accepted 9 September 1997) Abstract Rccombinant homoserine dehydrogenase from Saccharo- myces cerevisiae has been crystallized in three different forms. Crystals of the apo-cnzymc belong to the tetragonal space group P4 and have unit-cell-dimensions a = h = 130 and c = 240 A. The resolution limit for these crystals is 3.9 ,~.. Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD" have the tetragonal space group P412~2 or its cnantiomorph P43212. The unit-cell dimensions for these crystals arc a = h = 80.4 and c = 250.2 ,~, and the observed resolution limit is 2.2,~. Protein crystals grown in the presence of the product e-homoserine and the inert NAD* analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P2~ with unit-cell parameters a = 58.8, h -- 104.2, c = 120.7 A, fl = 91.9. This last crystal form has a diffraction limit of 2.7 A resolution. 1. Introduction The frequency and severity of fungal infections have drama- tically escalated over the last two decades (Sternberg, 1994). While ten years ago Gram negative bacteria were a major source of infectious diseases, in several medical centres this threat has now been replaced by pathogenic fungi (Graybill, 1995). Individuals with compromised immune systems (e.g. AIDS patients, individuals undergoing chemotherapy, organ- transplant recipients and burn victims) are especially suscep- tible to infections (see for example Bodey, 1993). Several factors have been implicated as a reason for the observed rise in fungal infections. In particular, the increase in the number of immuno-compromised individuals (partly caused by the spread of AIDS) and the development of drug resistance in fungi are both considered to have contributed signilicantly (Iwata, 1992; Sternberg, 1994: DeMuri & Hostetter, 1995; Graybill, 1995). The latter factor of emerging drug resistance in fungi clearly necessitates the development of new treatment methodologies against fungal infections (Sternberg, 1994). This is underscored by reports of fungal strains which are resistant to all commonly available antifungal agents (Wise et al., 1993). Exploitation of metabolic differences between species has been a successful strategy in the development of antimicrobial agents. One of the important metabolic differences between fungi and humans is amino-acid biosynthesis. For example, unlike humans, fungi are capable of synthesizing the essential amino acids threonine, isoleucine and methionine through the fungal aspartate pathway (Umbarger, 1978). Thcrefore, t Currentaddress: Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada. ~ 1998 International Union of Crystallography Printed in Great Britain - all rights reserved specific inhibitors of this pathway may be useful as fungicides. That this hypothesis holds true is illustrated by the antifungal natural product, (S)-2-amino-4-oxo-5-hydroxypentanoic acid, which has been shown to specifically inhibit homoserinc dehydrogenase, one of the enzymes in the aspartate pathway (Yamaki et al., 1990, 1992). Fungal homoserine dehydrogenase catalyses a critical common step in the pathway, namely the conversion of aspartate semi-aldehyde to L-homoserine, using the co-factor NAD(P)H. The enzyme from S. cerevisiae is 359 amino acids and is active as a dimer (Yamaki et al., 1990). Sequence analysis reveals that the enzyme likely possesses a structural motif known as the Rossmann fold which is common to many NAD(P)H binding proteins (Lesk, 1995). We have initiated protein crystallographic studies of homoscrine dehydrogenase from S. cerevisiae in order to investigate the structural requirements for inhibition of this enzyme by compounds such as the natural antifungal product (S)-2-amino-4-oxo-5-hydroxypcntanoic acid. It is hoped that these studies will provide information that can be used in the development of novel antimicrobial agents for the treatment of fungal infections. 2. Experimental procedures 2.1. Purification and crystallization of homoserine dehydro- genase. Homoserine dehydrogenase from S. cerevisiae was over- expressed in Escherichia coil and purified using procedures described elsewhere (Jacques et al., 1998). Prior to crystal- lization, the protein was concentrated to an optical density of 9.8 at 280 nm, corresponding to a protein concentration of approximately 10 mg ml '~, in either 10 mM HEPES (pH 8.5) or 10 mM Tris-HCl buffer (pH 8.5). Homogeneity was tested by Coomassie and silver-stained SDS-PAGE, as well as isoelectric focusing, all of which indicated that the protein was at least 99% pure. Crystallization experiments were performed using the hanging-drop vapour-diffusion method (McPherson, 1976). The reservoirs contained 1 ml of precipitant solution and the drops contained 5 Ial of a 1:1 mixture of protein solution and precipitant. Three different groups of experiments were carried out: crystallization of the apo-enzyme, crystallization of the protein in the presence of fivefold molar excess of the co-factor NAD', and crystallization of the protein in the presence of five fold molar excess of the product L-homoserine and the inert NAD + analogue 3-aminopyridine adenine dinucleotide. For the latter co-crystallization experiment, the product of the reaction catalysed by homoserine dehy- drogenase, L-homoserine, was used instead of the substrate, aspartate semi-aldehyde, since aspartate semi-aldehyde is an Acta Crystallographica Section D ISSN 0907-4449 ~. 1998