title: Mutational analysis of allylic substrate binding site of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.
abstract: Undecaprenyl diphosphate (UPP) synthase catalyzes the sequential cis-condensation of isopentenyl diphosphate (IPP) onto (E,E)-farnesyl diphosphate (FPP). In our previous reports on the Micrococcus luteus B-P 26 UPP synthase, we have shown that the conserved residues in the disordered region from Ser-74 to Val-85 is crucial for the binding of FPP and the catalytic function [Fujikura, K., et al. (2000) J. Biochem. (Tokyo) 128, 917-922] and the existence of a structural P-loop motif for the FPP binding site [Fujihashi, M., et al. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 4337-4342]. To elucidate the allylic substrate binding site in more detail, we prepared eight mutant enzymes and examined their kinetic behavior. The mutant with respect to the two complementarily conserved Arg residues among the structural P-loop motif, G32R-R42G, retained the activity and showed product distribution pattern exactly similar to that of the wild-type, indicating that the complementarily conserved Arg is important for maintaining the catalytic function. Substitutions of Asp-29, Arg-33, or Arg-80 with Ala resulted in a large loss of enzyme activity, suggesting that these residues are essential for catalytic function. However, the K(m) values of these mutant enzymes for Z-GGPP, which is the first intermediate during the enzymatic cis-condensations of IPP onto FPP, were only moderately different or little changed from those of the wild type. These results suggest that the binding site for the intermediate Z-GGPP having a cis double bond is different to that for the intrinsic allylic substrate, FPP, whose diphosphate moiety is recognized by the structural P-loop.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Fujikura
authors.forename: Yuan-Wei
authors.initials: YW
authors.lastname: Zhang
authors.forename: Masahiro
authors.initials: M
authors.lastname: Fujihashi
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
authors.forename: Tanetoshi
authors.initials: T
authors.lastname: Koyama
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002855:Chromatography, Thin Layer; D007700:Kinetics; D016982:Micrococcus luteus / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
chemical_list: D019883:Alkyl and Aryl Transferases; C023266:undecaprenyl pyrophosphate synthetase