Roles of Amino Acids in the Escherichia coli Octaprenyl Diphosphate Synthase Active Site Probed by Structure-Guided Site-Directed Mutagenesis

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• 作者：Keng-Ming Chang ; Shih-Hsun Chen ; Chih-Jung Kuo ; Chi-Kang Chang ; Rey-Ting Guo ; Jinn-Moon Yang ; Po-Huang Liang
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：April 24, 2012
• 年：2012
• 卷：51
• 期：16
• 页码：3412-3419
• 全文大小：449K
• 年卷期：v.51,no.16(April 24, 2012)
• ISSN：1520-4995

文摘

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 (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.