Structural characterization of the active flap of IMP dehydrogenase: A link between drug selectivity and catalytic efficiency

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the reduction of NAD. The reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis, which makes IMPDH a drug target in immunosuppressive, antiviral and antitumor chemotherapy.; Mycophenolic acid (MPA) is an IMPDH inhibitor widely used as an immunosuppressive agent in kidney transplantation. MPA specifically binds to the E-XMP* intermediate formed in the reaction pathway. Mammalian IMPDHs have higher affinity for MPA but lower kcat, while microbial isozymes have lower affinity for MPA but higher kcat. The selectivity of MPA is attributed to the accumulation of E-XMP* in the reaction, the affinity of the MPA binding site and an conformational change propagating from the nicotinamide subsite to the adenosine subsite of NAD site. A non-conserved distal flap in the active site appears to be important to the MPA affinity and IMPDH activity, so its movement may account for the unknown conformational change.; The goal of this thesis is to identify the structural basis of the conformational change that determines the MPA selectivity and the catalytic efficiency of IMPDH. We solved two x-ray crystal structures of the Tritrichomonas foetos IMPDH catalytic domain in complex with IMP and beta-Me-TAD and in complex with a transition state analog mizoribine 5'-monophosphate (MZP), respectively. The E·IMP·beta-Me-TAD structure defines the NAD site and reveals the structural difference of the adenosine end between microbial and mammalian IMPDHs. The binding of NAD also orients the active site loop for catalysis. However, the distal flap is disordered in the ternary complex. The E·MZP structure captures a closed conformation of the distal flap in the nicotinamide subsite, suggesting it competes for the same binding site with MPA. The conserved Arg-Tyr in the distal flap coordinates to MZP via a water molecule, suggesting it activates the water to hydrolyze the E-XMP* intermediate. Therefore, the distal flap controls both drug selectivity and catalytic efficiency of IMPDH by switching between the open and closed conformations.