The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the ␤A-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four ␤-strands (␤A-␤B-␤D-␤C) and two helices (␣2 and ␣3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C 70 polymer rather than the C 55 polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C 30 intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C 55 product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C 55 product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k cat values. W75A showed an 8-fold increase of the FPP K m value, and 22-33-fold increases in the IPP K m values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release. Prenyltransferases catalyze consecutive condensation reactions of isopentenyl pyrophosphate (IPP)1 with allylic pyrophosphate to generate linear isoprenyl polymers. The isoprenylates undergo further modification to form a variety of isoprenoid structures including steroids, terpenes, the side chains of respiratory quinones, carotenoids, natural rubber, the glycosyl carrier lipid, and prenyl proteins (1, 2). E-and Z-type prenyltransferases synthesize trans and cis double bonds, respectively, through the condensation reactions of IPP (3). Each of the E-type enzymes catalyzes the formation of a product having a specific chain length ranging from C 10 to C 50 (4).Two conserved DDXXD motifs are observed in E-type enzymes (5-7). X-ray structural (8) and site-directed mutagenesis studies (9 -12) of farnesyl-pyrophosphate synthase (FPPs) have shown that the first aspartate-rich motif binds the allylic substrate, whereas the second DDXXD binds IPP via Mg 2ϩ . Mutagenesis studies indicate that the 5th amino acid residue (Phe-77) upstream from the first DDXXD plays a critical role in controlling the chain length of the final product formed in the reaction catalyzed by E-type geranylgeranyl-pyrophosphate synthase from archaebacterium (13). By substituting this large residue with the smaller Ser, product synthesis was shifted from the production of C 20 product to C 25 and C 30 products (14). Double (F77...

Isoprenoids are an extensive group of natural products consisting of five-carbon isopentenyl units (1, 2). A class of enzymes involved in the biosynthesis of the linear isoprenoid polymers each catalyzes consecutive 1Ј-4 condensation reactions of a designated number of isopentenyl pyrophosphate (IPP) 1 with a single farnesyl pyrophosphate (FPP) (3). These prenyltransferases are classified as cis-and trans-isoprenyl pyrophosphate synthases according to the stereochemical outcome of their products resulted from IPP condensation (4). The enzymatic products play essential biological roles; for example, the trans-C 40 octaprenyl pyrophosphate (OPP) synthesized by trans-type OPP synthase constitutes the side chain of ubiquinone (5, 6), and the C 55 undecaprenyl pyrophosphate (UPP) synthesized by cis-type UPPs serves as a lipid carrier for bacterial peptidoglycan biosynthesis (7,8). Cis-and trans-prenyltransferases apparently utilize different strategies for substrate binding and catalysis while sharing the same allylic substrate FPP and homoallylic substrate IPP. This was initially supported by the lack of sequence similarity between the two groups of prenyltransferases (9, 10) and was further validated unequivocally by the crystal structures of both OPP synthase and UPPs (11-16). The trans-type enzymes involve a mechanism of ionization, condensation and elimination reactions, which are initiated by breaking the bond between the pyrophosphate and the farnesyl group, followed by electrophilic attack of the C1 carbonium of the farnesyl on the C4 of IPP, and concluded with elimination of the proton on C2 of IPP (17). The elimination of the pyrophosphate group of FPP is facilitated by the Mg 2ϩ that is coordinated to the DDXXD motif conserved in all the trans-prenyltransferases (18 -20).The reaction mechanism for catalysis of the cis-type enzymes is less understood. In the cis-type UPPs, no DDXXD motif was found, and our previous fluorescence binding study showed that FPP binding did not require Mg 2ϩ , whereas IPP binding and the ensuing reactions absolutely required the metal ion (21). Based on the crystal structure of UPPs in complex with FPP,

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. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the ␣3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asp-rich motifs for substrate binding via Mg 2+ . The new structure provides a plausible mechanism for the catalysis of UPPs.Keywords: prenyltransferase; farnesyl pyrophosphate; isopentenyl pyrophosphate; crystal structure; substrate binding; metal ion (Sato et al. 1999;Chang et al. 2001). Rubber prenyltransferase synthesizes a huge polymer containing thousands of IPP units (Cornish 2001). In contrast, trans-prenyltransferases, which generate trans-double bonds in IPP condensation, synthesize shorter chain-length products, C 15 -C 50 (Chen et al. 1994; Wang and Ohuma 2000). Abbreviations: UPPs, undecaprenyl pyrophosphate synthase; IPP, isopentenyl pyrophosphate; FPP, farnesyl pyrophosphate; UPP, undecaprenyl pyrophosphate; FPPs, farnesyl pyrophosphate synthase; GPP, geranyl pyrophosphate; TLC, thin-layer chromatography; NiNTA, nickel nitrilo-triacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; FTase, farnesyltransferase.Article and publication are at http://www.proteinscience.org/cgi

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...