Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis.
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the reduction of NAD(+), which is the rate-limiting step in the biosynthesis of guanine nucleotides. IMPDH is a promising target for chemotherapy. Microbial IMPDHs differ from mammalian enzymes in their lower affinity for inhibitors such as mycophenolic acid (MPA) and thiazole-4-carboxamide adenine dinucleotide (TAD). Part of this resistance is determined by the coupling between nicotinamide and adenosine subsites in the NAD(+) binding site that is postulated to involve an active site flap. To understand the structural basis of the drug selectivity, we solved the X-ray crystal structure of the catalytic core domain of Tritrichomonas foetus IMPDH in complex with IMP and beta-methylene-TAD at 2.2 A resolution. Unlike previous structures of this enzyme, the active site loop is ordered in this complex, and the catalytic Cys319 is 3.6 A from IMP, in the same plane as the hypoxanthine ring. The active site loop forms hydrogen bonds to the carboxamide of beta-Me-TAD which suggests that NAD(+) promotes the nucleophillic attack of Cys319 on IMP. The interactions of the adenosine end of TAD are very different from those in the human enzyme, suggesting the NAD(+) site may be an exploitable target for the design of antimicrobial drugs. In addition, a new K(+) site is observed at the subunit interface. This site is adjacent to beta-Me-TAD, consistent with the link between the K(+) activation and NAD(+). However, contrary to the coupling model, the flap does not cover the adenosine subsite and remains largely disordered.
Gan
Lu
L
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Petsko
Gregory A
GA
Hedstrom
Lizbeth
L
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002414:Cations, Monovalent / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000235:genetics; D020536:Enzyme Activators / Q000737:chemistry; D006860:Hydrogen Bonding; D007168:IMP Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007291:Inosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry*; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry; D016835:Tritrichomonas foetus / Q000201:enzymology* / Q000235:genetics
D002414:Cations, Monovalent; D020536:Enzyme Activators; D046911:Macromolecular Substances; D015800:Protozoan Proteins; D013844:Thiazoles; D009243:NAD; C085445:beta-methylene thiazole-4-carboxamide adenine dinucleotide; D007291:Inosine Monophosphate; D000244:Adenosine Diphosphate; D007168:IMP Dehydrogenase; D011188:Potassium