Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C-55 product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-H-2(2)]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 +/- 0.022 measured for UPPS reaction using [1,1-2H2]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism. (C) 2010 Elsevier Inc. All rights reserved.