Roles of amino acids in the Escherichia coli octaprenyl diphosphate synthase active site probed by structure-guided site-directed mutagenesis

Keng Ming Chang, Shih Hsun Chen, Chih Jung Kuo, Chi Kang Chang, Rey Ting Guo, Jinn-Moon Yang, Po Huang Liang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Octaprenyl diphosphate synthase (OPPS) catalyzes consecutive condensation reactions of farnesyl diphosphate (FPP) with five molecules of isopentenyl diphosphates (IPP) to generate C40 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 (MgPPi) for HR 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.

Original languageEnglish
Pages (from-to)3412-3419
Number of pages8
JournalBiochemistry
Volume51
Issue number16
DOIs
StatePublished - 24 Apr 2012

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