Characteristics and Crystal Structure of Bacterial Inosine-5′-monophosphate Dehydrogenase †,‡ Rong-guang Zhang, Gwyndaf Evans, Frank J. Rotella, Edwin M. Westbrook, Don Beno, Eliezer Huberman, Andrzej Joachimiak, and Frank R. Collart* Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 South Cass AVenue, Argonne, Illinois 60439-4833 ReceiVed December 3, 1998; ReVised Manuscript ReceiVed January 28, 1999 ABSTRACT: IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the K m for NAD (1180 µM) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 Å with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne’s Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione -synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors. Inosine monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) 1 is a rate-limiting enzyme in the synthesis of guanine ribonucleotides. IMPDH has an essential role in providing critical precursors for DNA and RNA biosynthesis and in signal transduction pathways that mediate cell differentiation (1, 2). This essential nature is illustrated by the utility of IMPDH inhibitors as therapeutic agents. Several potent inhibitors of mammalian IMPDH enzymes are used clinically as antiviral, anticancer, or immunosuppressive agents (3-5). However, the utility of IMPDH inhibitors as antimicrobial agents has not been fully investigated. Sequence analysis of all known IMPDH enzymes supports a distinction between the bacterial and eukaryotic enzymes. A deep branching of the bacterial and eukaryotic forms of IMPDH is observed upon phylogenetic analysis of the relationships among the various IMPDH genes (6, 7). The analysis indicates a general functional conservation of amino acid residues and suggests a unique amino acid sequence signature for these kingdoms. The phylogenetic differences between IMPDH enzymes reflect their kinetic differences and differential sensitivity to inhibitors. Enzymes from mammalian sources show distinctly lower values for the K m for NAD than do those enzymes from bacteria (8-10). In addition, mammalian IMPDH enzymes are several orders of magnitude more sensitive to inhibition by mycophenolic acid (MPA) than are bacterial IMPDH enzymes (8-10). We hypothesize that the biochemical and kinetic differences between bacterial and mammalian enzymes are a conse- quence of the variance of specific, identifiable amino acid residues. Identification of the critical residues or combination of residues is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. IMPDH catalyzes the NAD-dependent oxidation of IMP to XMP, a primary step in the biosynthesis of guanine ribonucleotides. The reaction mechanism has been examined in detail for IMPDH from Escherichia coli (11), Tritri- chomonas foetus (12), and humans (13). These enzymes use a sequential, ordered, bi-bi kinetic mechanism in which IMP binds before NAD and NADH is released before XMP † This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research, under Contract W-31-109- ENG-38. ‡ Coordinates and structure factors have been deposited in the Brookhaven Protein Data Bank (PDB) under ID code 1ZFJ. * Correspondence should be addressed to this author. E-mail: Fcollart@anl.gov. Phone: 630-252-4859. Fax: 630-252-3387. 1 Abbreviations: IMPDH, IMP dehydrogenase; GTP, guanosine triphosphate; IMP, inosine monophosphate; XMP, xanthosine mono- phosphate; MALDI-MS, matrix-assisted laser desorption ionization mass spectroscopy; MAD, multiwavelength anomalous diffraction; MPA, mycophenolic acid; PDB, Protein Data Bank; AU, asymmetric unit; NAD, nicotinamide adenine dinucleotide; MW, molecular weight; SeMet, selenomethionine; CBS, cystathionine -synthase; FOM, figure of merit; IPTG, isopropylthiogalactopyranoside. 4691 Biochemistry 1999, 38, 4691-4700 10.1021/bi982858v CCC: $18.00 © 1999 American Chemical Society Published on Web 03/26/1999