Catalytic center assembly of HPPK as revealed by the crystal structure of a ternary complex at 1.25 angstrom resolution

Background: Folates are essential for life. Unlike mammals, most microorgan isms must synthesize folates de novo. 6-Hydroxymethyl-7,8-dihydropterin pyr ophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydro xymethyl-7,8-dihydropterin (HP), the first reaction in the folate pathway, and therefore is an ideal target for developing novel antimicrobial agents. HPPK from Escherichia coil is 158-residue thermostable protein that provid es a convenient model system for mechanistic studies. Crystal structures ha ve been reported for HPPK without bound ligand, containing an HP analog, an d complexed with an HF analog, two Mg2+ ions, and ATP. Results: We present the 1.25 Angstrom crystal structure of HPPK in complex with HP, two Mg2+ ions, and AMPCPP (an ATP analog that inhibits the enzymat ic reaction). This structure demonstrates that the enzyme seals the active center where the reaction occurs. The comparison with unligated HPPK reveal s dramatic conformational changes of three flexible loops and many sidechai ns. The coordination of Mg2+ ions has been defined and the roles of 26 resi dues have been derived. Conclusions: HPPK-HP-MgAMPCPP mimics most closely the natural ternary compl ex of HPPK and provides details of protein-substrate interactions. The coor dination of the two Mg2+ ions helps create the correct geometry for the one -step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transi tion state. The rigidity of the adenine-binding pocket and hydrogen bonds a re responsible for adenosine specificity. The nonconserved residues that in teract with the substrate might be responsible for the species-dependent pr operties of an isozyme.