This proposal seeks to exploit the significant advances made in structurally characterizing rat dihydropteridine reductase (DHPR) during the last support period. Two crystal forms of the enzyme were obtained with resolution less than 2 Angstroms and structures of the apo enzyme and a binary complex with NADH were characterized. In addition, the DHPR gene was cloned and expressed in Escherichia coli and successful mutagenesis experiments were performed. With this foundation it is intended to generate specific mutants to define the mechanism of enzymatic action and confirm discovered similarities to flavin enzyme mechanisms and to those of the short-chain dehydrogenases. All mutants will be characterized for stability, specific activity and kinetics. Attempts will be made to create a ternary complex by: (a) using molecular graphics; (b) specific mutants having substrate affinity but no activity, and; (c) crystal soaks with the recently resolved monoclinic crystal form that contains a more solvated molecular structure than the orthorhombic form that has earlier proven intransigent to this approach. The human DHPR cDNA gene sequence is closely related to the rat and will be generated either by mutagenic construction, or by PCR techniques from a lambda phage library and the expressed protein crystal structure will be rapidly obtained by molecular replacement methodology. It was recently observed that treatment of DHPR with the active fraction from cAMP kinase gives uptake of one phosphate/subunit. The structural effects will be delineated by crystallography and mechanistic effects by kinetic analyses. An active monomer will be created by mutating specific amino acids that contribute to the amphipathic hydrophobic four helix bundle that holds the two monomers in dimeric form. This will allow multidimensional NMR techniques to be used to assist in mechanistic analysis. It is intended to determine and correlate naturally occurring genetic errors from patients with aberrant PKU with the known structure and attempt to discover patterns of mechanistic and structural disruption. The errors will also be reproduced in the E. coli expression system for in vitro examination of enzyme action. It is also intended to elaborate the mutant picture to determine the key functional regions for viable activity. By applying graphics analysis to the perceived active site, it is intended to select an inhibitor with DHPR specificity as none yet exist. It is also intended to explore further our initial intriguing experiments to deliver DHPR to the cytosol via protein-bound folate and the folate binding protein of the cellular membrane. Two aspects of the superficially similar enzyme dihydrofolate reductase (DHFR) are also to be explored. In one instance the 'aptamer' technique will be employed to detect the known distinction that exists between the antifolate inhibitor sites of prokaryotic and eukaryotic sources of this enzyme as a model for proving the feasibility of aptamer technology, and secondly the neglected field of mycobacterial DHFRs will be probed by the isolation and structural and mechanistic characterization of this enzyme from Mycobacterium tuberculosis and Mycobacterium avium.
Showing the most recent 10 out of 24 publications
