Probing the Conformational Change of Escherichia coliUndecaprenyl Pyrophosphate Synthase during Catalysis Using an Inhibitor and Tryptophan Mutants

Abstract

Undecaprenyl pyrophosphate synthase (UPPS)1 catalyzes the chain elongation of farnesyl pyrophosphate (FPP) with eight molecules of isopentenyl pyrophosphate (IPP) to generate C 55 undecaprenyl pyrophosphate (UPP) (1-4). It belongs to a cis-prenyltransferase family and shows sequence homology with other members including dehydrodolichyl pyrophosphate synthase from yeast (5), a C 15 and a C 50 isopentenyl pyrophosphate synthases found in Mycobacterium tuberculosis (6), and a C 120 prenyltransferase from Arabidopsis thaliana (7). The biological function of bacterial UPP is to serve as a carrier to transport UDP-N-acetylmuramic acid pentapeptide, which subsequently reacts with UDP-N-acetylglucosamine to form lipid II, to extracellular compartments where lipid II molecules are assembled into peptidoglycans of the cell wall (8, 9). The enzyme is essential for bacterial survival due to its vital role in cell wall biosynthesis (10). The crystal structures of UPPS from Micrococcus luteus and Escherichia coli solved, respectively, by Fujihashi et al. (11) and by Ko et al. (12) are the first two structures in the cis-prenyltransferase family. In E. coli UPPS structure, an elongated tunnel with the hydrophobic amino acid side chains covering the entire interior surface was proposed to accommodate the UPP product (Fig. 1A) (12). This hydrophobic tunnel is surrounded by two ␣-helices (␣2 and ␣3) and four ␤-strands (␤A-␤B-␤D-␤C). On the top portion of the tunnel, several conserved hydrophilic amino acids in the vicinity of Asp-26 including Asn-28, Arg-30, His-43, Phe-70, Ser-71, in subunit A as well as the Glu-213 from subunit B of the UPPS homodimer are proposed to involve in the FPP and IPP binding. Site-directed mutagenesis studies suggest that IPP is probably bound to the area of since mutations of these residues to Ala resulted in significantly increased IPP K m and decreased k cat values (13-15). FPP is proposed to bind with its pyrophosphate group near the area and its C 15 -tail pointing toward the bottom of the active site (11,12,14,15).A disordered loop consisting of the amino acid residues 72-83 is proposed to bring the IPP to a correct position and orientation relative to FPP, needed to initiate the condensation reaction (12). The replacement of Trp-75 residue, located in the loop with Ala, increases the FPP and IPP K m values, indicating that Trp-75 is close to the bound IPP and FPP (12,15). The mutations of several other residues such as N74A, E81A, and R77A of the loop display decreased k cat and increased IPP K m values (12). The loop may also serve as a hinge for the necessary protein conformational change in catalysis since two conformers observed in the E. coli UPPS structure differ in the position of ␣3 helix (Fig. 1A), which is connected to the loop (12). In the closed conformer, the tunnel is narrow within which an elongated electron density envelope (probably Triton or polyethylene glycol used in the crystallization) is visible. On the other hand, the open form of the tunnel contains only water mole...

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

“…Regarding the role of Mg 2ϩ in substrate binding, our fluorescence studies showed that the FPP substrate still binds to UPPs and quenches its intrinsic fluorescence even in the absence of Mg 2ϩ (17). However, the IPP binding absolutely requires Mg 2ϩ .…”

“…The interconversion between two conformations of open and closed forms is implicated in substrate binding and product release. The conformational change during substrate binding and catalysis of UPPs has been probed previously using steady-state and stopped-flow fluorometers (17). It was shown that FPP binding quenches the fluorescence of Trp-91 in the ␣3 helix, which moves toward the active site during substrate binding and thereby results in a closed conformation to provide better interaction of UPPs with the substrate.…”

“…These two conformers have the most striking difference in the position of the ␣3 helix, which is connected to a loop containing amino acids 72-82. In the proposed catalytic model, the loop may play an essential role in pulling the ␣3 helix toward the active site (17). Perhaps because of the requirement of its flexibility in enzyme catalysis, this loop was invisible in the previous crystal structure of the apoenzyme.…”

Undecaprenyl Pyrophosphate Involvement in Susceptibility of Bacillus subtilis to Rare Earth Elements

“…3A), the uppS86 mutation appears to be a reduction-of-function mutation. Based on the results of previous structural and mutational analyses of E. coli C 55 -PP synthase (6,7,10,16), residue Thr86 (corresponding to Ser72 in E. coli C 55 -PP synthase) is located in the flexible loop, which is important for catalysis and substrate binding. Deletion of this residue in E. coli C 55 -PP synthase decreases the k cat value by 10 5 -fold (6).…”

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases

“…Medium-chain cis-PTs are represented by UPPS purified from a number of bacterial species (5,16,(23)(24)(25)(26)(27)(28)(29)(30), Z,E-mixed decaprenyl diphosphate synthase (Z,E-DecPP, C 50 ) from M. tuberculosis (Rv2361), as well as plant, protozoan, and archaeal enzymes. UPPS is responsible for the biogenesis of undecaprenyl phosphate, an indispensable glycosyl carrier lipid in bacterial cell wall biosynthesis.…”