W0473

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Because the folate pathway is essential for microorganisms but absent from mammals, HPPK and other enzymes in the pathway are ideal targets for developing antimicrobial agents. To elucidate the catalytic mechanism of HPPK, we have mapped out the reaction trajectory by determining the crystal and/or NMR solution structures at every catalytic stage, including apo-HPPK, binary complexes with either a substrate or a product, ternary complexes with either both substrates or both products, and a ternary complex that mimics the transition state. All crystal structures are determined at atomic resolution (up to 0.89 Å). Comparative analysis of these structures revealed that the complete active center of HPPK is assembled only upon the binding of both substrates (Blaszczyk et al. 2003. Biochemistry 42:1573-1580, and references cited there). The assembly of active center involves large substrate-induced conformational changes. HPPK has three catalytic loops, among which loop 3 (residues 81-93) undergoes most dramatic and unusual conformational changes. To investigate the catalytic role of loop 3, we have made a loop 3 deletion mutant and investigated its property by biochemical and X-ray crystallographic analysis. Three crystal structures of the mutant, including a ligand-free form and two forms of ternary complex with both substrates, have been determined at high resolution (1.48-1.33 Å). The ligand-free structure superimposes well with that of the wild type, indicating that the deletion mutation does not affect protein folding. Although the core of the two complex structures is the same as that of the wild type, they do not have a fully assembled active center. The results suggest that loop 3 is required for the assembling of the full active center and is therefore critical for catalysis.