Characteristics and crystal structure of bacterial inosine-5 '-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first s tep unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IM PDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are s imilar to other bacterial IMPDH enzymes. However, the lack of sensitivity t o mycophenolic acid and the K-m for NAD (1180 mu M) exemplify some of the d ifferences 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 a t 1.9 Angstrom with substrate bound in the catalytic site. The structure wa s 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 subu nits 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 beta-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 d ifferences in the catalytic environment of bacterial and mammalian IMPDH en zymes. Comparison of the structure of bacterial IMPDH with the known partia l structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMP DH inhibitors.