title: Mutagenesis of isopentenyl phosphate kinase to enhance geranyl phosphate kinase activity.
abstract: Isopentenyl phosphate kinase (IPK) catalyzes the ATP-dependent phosphorylation of isopentenyl phosphate (IP) to form isopentenyl diphosphate (IPP) during biosynthesis of isoprenoid metabolites in Archaea. The structure of IPK from the archeaon Thermoplasma acidophilum (THA) was recently reported and guided the reconstruction of the IP binding site to accommodate the longer chain isoprenoid monophosphates geranyl phosphate (GP) and farnesyl phosphate (FP). We created four mutants of THA IPK with different combinations of alanine substitutions for Tyr70, Val73, Val130, and Ile140, amino acids with bulky side chains that limited the size of the side chain of the isoprenoid phosphate substrate that could be accommodated in the active site. The mutants had substantially increased GP kinase activity, with 20-200-fold increases in k(cat)(GP) and 30-130-fold increases in k(cat)(GP)/K(M)(GP) relative to those of wild-type THA IPK. The mutations also resulted in a 10(6)-fold decrease in k(cat)(IP)/K(M)(IP) compared to that of wild-type IPK. No significant change in the kinetic parameters for the cosubstrate ATP was observed, signifying that binding between the nucleotide binding site and the IP binding site was not cooperative. The shift in substrate selectivity from IP to GP, and to a lesser extent, FP, in the mutants could act as a starting point for the creation of more efficient GP or FP kinases whose products could be exploited for the chemoenzymatic synthesis of radiolabeled isoprenoid diphosphates.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, 84112, United States.
authors.forename: Mark F
authors.initials: MF
authors.lastname: Mabanglo
authors.forename: Jian-Jung
authors.initials: JJ
authors.lastname: Pan
authors.forename: Binita
authors.initials: B
authors.lastname: Shakya
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D004789:Enzyme Activation / Q000502:physiology; D007700:Kinetics; D016296:Mutagenesis / Q000502:physiology*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D011494:Protein Kinases; C402428:isopentenyl monophosphate kinase