Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time.

Farnesyl pyrophosphate (FPP) is a common substrate for a variety of prenyltransferases for synthesizing isoprenoid compounds. In this study, (2E,6E)-8-O-(N-methyl-2-aminobenzoyl)-3,7-dimethyl-2,6-octandien-1-pyrophosphate (MANT-O-GPP), a fluorescent analog of FPP, was synthesized and demonstrated as a satisfactory substrate for Escherichia coli undecaprenyl pyrophosphate synthase (UPPS) with a K(m) of 1.5 μM and a k(cat) of 1.

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 Mg(2+)/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 Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site.

Substrate and product specificities of cis-type undecaprenyl pyrophosphate synthase.

UPPS (undecaprenyl pyrophosphate synthase) catalyses consecutive condensation reactions of FPP (farnesyl pyrophosphate) with eight isopentenyl pyrophosphates to generate C55 UPP, which serves as a lipid carrier for bacterial peptidoglycan biosynthesis. We reported the co-crystal structure of Escherichia coli UPPS in complex with FPP. Its phosphate head-group is bound to positively charged arginine residues and the hydrocarbon moiety interacts with hydrophobic amino acids including L85, L88 and F89, located on the alpha3 helix of UPPS.

Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg(2+), and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved.

Synthesis and application of a fluorescent substrate analogue to study ligand interactions for undecaprenyl pyrophosphate synthase.

Farnesyl pyrophosphate (FPP) serves as a common substrate for many prenyltransferases involved in the biosynthesis of isoprenoid compounds. Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the chain elongation of FPP to C(55) undecaprenyl pyrophosphate (UPP) which acts as a lipid carrier in bacterial peptidoglycan synthesis. In this study, 7-(2,6-dimethyl-8-diphospho-2,6-octadienyloxy)-8-methyl-4-trifluoromethyl-chromen-2-one geranyl pyrophosphate, a fluorescent analogue of FPP, was prepared and utilized to study ligand interactions with E.

Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants.

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate (UPP). In the present study, site-directed mutagenesis, fluorescence quenching, and stopped-flow methods were utilized to examine the substrate binding and the protein conformational change. (S)-Farnesyl thiopyrophosphate (FsPP), a FPP analogue, was synthesized to probe the enzyme inhibition and events associated with the protein fluorescence change.