Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima: how is it different from that of the mesophilic enzyme

Abstract

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all- trans C 40-octaprenyl pyrophosphate. The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP. Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s −1 but product release limits the steady-state rate at 0.02 s −1 [Biochim. Biophys. Acta 1594 (2002) 64]. In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined. The enzyme activity at 25 °C was 0.005 s −1 under steady-state condition and was exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction. The product of chain elongation catalyzed by T. maritima OPPs was C 40 and the rate of its conversion to C 45 was negligible. Under single-turnover condition with 10 μM OPPs·FPP complex and 1 μM IPP, only the C 20 was formed rather than C 20–C 40 observed for E. coli enzyme. Together, our data suggest that the thermophilic OPPs from T. maritima has lower enzyme activity at 25 °C, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E. coli.