title: Roles of amino acids in the Escherichia coli octaprenyl diphosphate synthase active site probed by structure-guided site-directed mutagenesis.
abstract: Octaprenyl diphosphate synthase (OPPS) catalyzes consecutive condensation reactions of farnesyl diphosphate (FPP) with five molecules of isopentenyl diphosphates (IPP) to generate C(40) octaprenyl diphosphate, which constitutes the side chain of ubiquinone or menaquinone. To understand the roles of active site amino acids in substrate binding and catalysis, we conducted site-directed mutagenesis studies with Escherichia coli OPPS. In conclusion, D85 is the most important residue in the first DDXXD motif for both FPP and IPP binding through an H-bond network involving R93 and R94, respectively, whereas R94, K45, R48, and H77 are responsible for IPP binding by providing H-bonds and ionic interactions. K170 and T171 may stabilize the farnesyl carbocation intermediate to facilitate the reaction, whereas R93 and K225 may stabilize the catalytic base (MgPP(i)) for H(R) proton abstraction after IPP condensation. K225 and K235 in a flexible loop may interact with FPP when the enzyme becomes a closed conformation, which is therefore crucial for catalysis. Q208 is near the hydrophobic part of IPP and is important for IPP binding and catalysis.
authors.affiliation: Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
authors.forename: Keng-Ming
authors.initials: KM
authors.lastname: Chang
authors.forename: Shih-Hsun
authors.initials: SH
authors.lastname: Chen
authors.forename: Chih-Jung
authors.initials: CJ
authors.lastname: Kuo
authors.forename: Chi-Kang
authors.initials: CK
authors.lastname: Chang
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
authors.forename: Jinn-Moon
authors.initials: JM
authors.lastname: Yang
authors.forename: Po-Huang
authors.initials: PH
authors.lastname: Liang
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D011756:Diphosphates; D019883:Alkyl and Aryl Transferases; C048987:octaprenyl pyrophosphate synthetase