Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: Roles of the metal ion and conserved residues in catalysis

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg²⁺/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg²⁺/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg2+ is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp²⁶, and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg²⁺] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg²⁺] = 1 mM but drops significantly when Mg²⁺ ions are in excess (50 mM). Without Mg²⁺, IPP binds to UPPs only at high concentration. Mutation of Asp²⁶ to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp²⁶ is probably to assist the migration of Mg²⁺ from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His⁴³, Ser⁷¹, Asn⁷⁴, and Arg⁷⁷, may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.