Catalytic Center Assembly of HPPK as Revealed by the Crystal Structure of a Ternary Complex at 1.25 Å Resolution

摘要: Abstract Background: Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in folate pathway, and therefore is an ideal target developing novel antimicrobial agents. HPPK Escherichia coli a 158-residue thermostable protein that provides convenient model system mechanistic studies. Crystal structures have been reported without bound ligand, containing HP analog, complexed with two Mg 2+ ions, ATP. Results: We present 1.25 A crystal structure of complex HP, AMPCPP (an analog inhibits enzymatic reaction). This demonstrates enzyme seals active center where occurs. The comparison unligated reveals dramatic conformational changes three flexible loops many sidechains. coordination ions has defined roles 26 residues derived. Conclusions: HPPK–HP–MgAMPCPP mimics closely natural ternary details protein–substrate interactions. helps create correct geometry one-step transfer, which we suggest in-line single displacement mechanism some associative character transition state. rigidity adenine-binding pocket hydrogen bonds responsible adenosine specificity. nonconserved interact substrate might be species-dependent properties isozyme.