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ABSTRACT

Catalytic Mechanism Revealed by the Crystal Structure of Undecaprenyl
Pyrophosphate Synthase in Complex with Sulfate, Magnesium and Triton

Tzu-Ping Ko*, Sing-Yang Chang, Po-Huang Liang, and Andrew H.-J. Wang
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
128 Academia Road Section 2, Nankang, Taipei 11529, Taiwan

        Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP). UPP is a lipid carrier to mediate bacterial peptidoglycan synthesis. UPPs from Escherichia coli is a dimer and each subunit consists of 253 amino-acid residues. The chain length of the product is modulated by a hydrophobic active-site tunnel surrounded by two a-helices and four b-strands. New crystals of E. coli UPPs were grown in the presence of ammonium sulfate, magnesium chloride and Triton X-100, but turned out to be isomorphous to the previously solved apo-enzyme crystals. The crystals belong to the orthorhombic space group of P212121 with unit-cell dimension of a = 63.96 Å, b = 67.42 Å, c = 111.76 Å. The structure was refined to 1.73 Å resolution with R and Rfree values of 0.176 and 0.208, respectively. It showed bound sulfate and magnesium ions, as well as Triton X-100 molecules. The amino acid residues 72 – 82, which encompass an essential catalytic loop not seen in the previously determined structure, also became visible in one subunit. The sulfate ions suggest locations of the pyrophosphate groups of FPP and IPP in the active site. The site for binding FPP corresponds to a highly conserved structural P-loop in the enzyme. The Mg2+ is chelated by His199 and Glu213 from different subunits and possibly plays a structural rather than catalytic role. However, the metal ion is near the IPP-binding site and double mutation of His199 and Glu213 to alanines showed a remarkable increase of Km value for IPP. Inside the tunnel, one Triton surrounds the top portion of the tunnel and the other occupies the bottom part. These two Triton molecules may mimic the hydrocarbon moiety of the UPP product in the active site, with the cis-isoprenyl groups on the top and the all-trans farnesyl part in the bottom. Based on these observations, a possible reaction mechanism for UPPs catalysis is proposed, in which the side chains of Asp26 and His43 play the key roles of general base and general acid, respectively, to deprotonate and protonate the substrate molecules.

Institute of Biological Chemistry Academia Sinica
Contact :
biophys@gate.sinica.edu.tw