Roles of Amino Acids in the Escherichia coli Octaprenyl Diphosphate Synthase Active Site Probed by Structure-Guided Site-Directed Mutagenesis
Resource
BIOCHEMISTRY, 51(16), 3412-3419
Journal
BIOCHEMISTRY
Journal Volume
51
Journal Issue
16
Pages
3412-3419
Date Issued
2012
Date
2012
Author(s)
Chang, Keng-Ming
Chen, Shih-Hsun
Kuo, Chih-Jung
Chang, Chi-Kang
Guo, Rey-Ting
Yang, Jinn-Moon
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. © 2012 American Chemical Society.
Type
journal article
File(s)![Thumbnail Image]()
Loading...
Name
15.pdf
Size
23.17 KB
Format
Adobe PDF
Checksum
(MD5):6739c716ca3c89e65f4b353cabe5818d