proteins STRUCTURE O FUNCTION O BIOINFORMATICS STRUCTURE NOTE Crystal structure of a 3-oxoacyl-(acyl carrier protein) reductase (BA3989) from Bacillus anthracis at 2.4-A ˚ resolution Nathan R. Zaccai, 1 Lester G. Carter, 1 Nick S. Berrow, 1 Sarah Sainsbury, 1 Joanne E. Nettleship, 1 Thomas S. Walter, 1 Karl Harlos, 1 Ray J. Owens, 1 Keith S. Wilson, 2 David I. Stuart, 1 and Robert M. Esnouf 1 * 1 The Oxford Protein Production Facility, Division of Structural Biology, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom 2 York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom Key words: b-ketoacyl-(acyl carrier protein)reductase; short-chain dehydrogenase/reductase (SDR); FabG; fatty acid biosynthesis; X-ray crystallography. INTRODUCTION The Oxford Protein Production Facility (OPPF) was established to develop methods for high-throughput pro- tein production and crystallization. As part of these developments, and in the context of the European SPINE project, a pilot study was undertaken on 48 proteins from Bacillus anthracis from protein families which were well conserved across a wide range of bacteria. 1,2 Bacillus anthracis, the causative agent of anthrax, is a large, gram- positive, spore-bearing bacterium. The genome of the Ames strain of the bacterium has been sequenced 3 show- ing two plasmids, pXO1 and pXO2, that carry the major virulence factors, as well as normal chromosomal DNA of 5.23 megabases, predicted to code for about 5311 genes. The set of 48 proteins chosen for study 1 are all encoded for by the chromosomal DNA. Annotation of the B. anthracis genome suggests it enc- odes for four or five 3-oxoacyl-[acyl carrier protein (ACP)] reductases. These form part of the b-ketoacyl- (ACP) reductase family, itself part of the short-chain de- hydrogenase/reductase (SDR) superfamily, whose mem- bers catalyze a broad range of reactions using nucleotide cofactors. This study reports the structure of gene prod- uct BA3989, a 3-oxoacyl-(ACP) reductase of 246 residues, determined to a resolution of 2.4 A ˚ using protein pro- duced by the high-throughput pipeline of the OPPF. This enzyme performs the first reductive step in de novo fatty- acid biosynthesis 4 : the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of ACP (Fig. F1 1). Fatty-acid biosynthesis occurs by a series of universal biochemical transformations that are critical to almost all cells, but the pathway followed by bacterial systems differs substan- tially from that of higher organisms. Since this pathway is predicted to be essential 5 it has been suggested as an attractive target for the development of novel antibiot- ics. 6–8 Although to date no antibiotics target 3-oxoacyl- (ACP) reductases, a possible lead compound, epigalloca- techin gallate from green tea, has been shown to be a potent inhibitor of the Escherichia coli oxoacyl reductase FabG. 9 MATERIALS AND METHODS Cloning, expression and purification followed standard OPPF pipeline protocols. 2,10,11 Briefly, the expression J_ID: Z7E Customer A_ID: 00181-2007 Cadmus Art: PROT 21624 Date: 14-AUGUST-07 Stage: I Page: 1 ID: ananda Date: 14/8/07 Time: 12:50 Path: J:/Production/PROT/VOL00000/070340/3B2/C2PROT070340 Grant sponsor: UK Medical Research Council, MRC; Grant sponsor: SPINE; Grant number: QLG2-CT-2002-00988. AQ2 Nathan R. Zaccai’s current address is Department of Pharmacology, University of Bristol, University Walk, Bristol BS8 1TD, UK. Lester G. Carter’s current address is Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St Andrews, KY16 9ST, UK. *Correspondence to: Dr. Robert Esnouf, Division of Structural Biology, University of Oxford, Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Oxford, OX3 7BN, UK. E-mail: robert@strubi.ox.ac.uk Received 13 March 2007; Accepted 26 March 2007 Published online 00 Month 2007 in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/prot.21624 V V C 2007 WILEY-LISS, INC. PROTEINS 1