The crystal structure of dihydrofolate reductase from Thermotoga maritima:Molecular features of thermostability

Two high-resolution structures have been obtained for dihydrofolate reducta se from the hyperthermophilic bacterium Thermotoga maritima in its unligand ed state, and in its ternary complex with the cofactor NADPH and the inhibi tor, methotrexate. While the overall fold of the hyperthermophilic enzyme i s closely similar to monomeric mesophilic dihydrofolate reductase molecules , its quaternary structure is exceptional, in that T. maritima dihydrofolat e reductase forms a highly stable homodimer. Here, the molecular reasons fo r the high intrinsic stability of the enzyme are elaborated and put in cont ext with the available data on the physical parameters governing the foldin g reaction. The molecule is extremely rigid, even with respect to structura l changes during substrate binding and turnover. Subunit cooperativity can be excluded from structural and biochemical data. Major contributions to th e high intrinsic stability of the enzyme result from the formation of the d imer. Within the monomer, only subtle stabilizing interactions are detectab le, without clear evidence for any of the typical increments of thermal sta bilization commonly reported for hyperthermophilic proteins. The docking of the subunits is optimized with respect to high packing density in the dime r interface, additional salt-bridges and beta-sheets. The enzyme does not s how significant structural changes upon binding its coenzyme, NADPH, and th e inhibitor, methotrexate. The active-site loop, which is known to play an important role in catalysis in mesophilic dihydrofolate reductase molecules , is rearranged, participating in the association of the subunits; it no lo nger participates in catalysis. (C) 2000 Academic Press.