Structures,mechanisms and inhibitors of undecaprenyl diphosphate synthase: A cis-prenyltransferase for bacterial peptidoglycan biosynthesis

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.     

Mechanism of cis-prenyltransferase reaction probed by substrate analogues

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C₅₅ product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-²H₂]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 ± 0.022 measured for UPPS reaction using [1,1-²H₂]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.     

Different reaction mechanisms for cis- and trans-prenyltransferases

Octaprenyl diphosphate synthase (OPPs) and undecaprenyl diphosphate synthases (UPPs) catalyze consecutive condensation reactions of farnesyl diphosphate (FPP) with 5 and 8 isopentenyl diphosphate (IPP) to generate C₄₀ and C₅₅ products with trans- and cis-double bonds, respectively. In this study, we used IPP analogue, 3-bromo-3-butenyl diphosphate (Br-IPP), in conjunction with radiolabeled FPP, to probe the reaction mechanisms of the two prenyltransferases. Using this alternative substrate with electron-withdrawing bromo group at the C3 position to slow down the condensation step, trapping of farnesol in the OPPs reaction from radiolabeled FPP under basic condition was observed, consistent with a sequential mechanism. In contrast, UPPs reaction yielded no farnesyl carbocation intermediate under the same condition with radiolabeled FPP and Br-IPP, indicating a concerted mechanism. Our data demonstrate the different reaction mechanisms for cis- and tran-prenyltransferases although they share the same substrates.     

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 1.2 s⁻¹ based on [¹⁴C]IPP consumption. Interesting, we found that its emission fluorescence intensity at 420 nm increased remarkably during chain elongation, thereby useful for real-time monitoring kinetics of UPPS to yield a K_m of 1.1 μM and a k_cat of 1.0 s⁻¹, consistent with those measured using radiolabeled substrate. Using this assay, the IC₅₀ of a known UPPS inhibitor farnesyl thiopyrophosphate (FsPP) was confirmed. Our studies provide a convenient and environmentally friendly alternative for kinetics and inhibition studies on UPPS drug target.     

Substrate Specificities of Wild and Mutated Farnesyl Diphosphate Synthases from Bacillus Stearothermophilus with Artificial Substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH₃OGPP) as allylic substrate homologs.

The wild-type FPP synthase reaction of HOGPP (and CH₃OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation.

On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

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. This compound with a much less labile thiopyrophosphate shows K(i) value of 0.2 microm in the inhibition of Escherichia coli UPPS and serves as a poor substrate, with the k(cat) value (3.1 x 10(-7) s(-1)) 10(7) times smaller than using FPP as the substrate. Reduction of protein intrinsic fluorescence was observed upon addition of FPP (or FsPP) to the UPPS solution. Moreover, fluorescence studies carried out using W91F and other mutant UPPS with Trp replaced by Phe indicate that FPP binding mainly quenches the fluorescence of Trp-91, a residue in the alpha3 helix that moves toward the active site during substrate binding. Using stopped-flow apparatus, a three-phase protein fluorescence change with time was observed by mixing the E.FPP complex with IPP in the presence of Mg(2+). However, during the binding of E.FsPP with IPP, only the fastest phase was observed. These results suggest that the first phase is due to the IPP binding to E.FPP complex, and the other two slow phases are originated from the protein conformational change. The two slow phases coincide with the time course of FPP chain elongation from C(15) to C(55) and product release.     

Partial purification and characterization of the short-chain prenyltransferases, gernayl diphospate synthase and farnesyl diphosphate synthase, from Abies grandis (grand fir)

In the conifer Abies grandis (grand fir), a secreted oleoresin rich in mono-, sesqui-, and diterpenes serves as a constitutive and induced defense against insects and pathogenic fungi. Geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) synthase, two enzymes which form the principal precursors of the oleoresin mono- and sesquiterpenes, were isolated from the stems of 2-year-old grand fir saplings. These enzymes were partially purified by sequential chromatography on DEAE-Sepharose, Mono-Q, and phenyl-Sepharose to remove competing phosphohydrolase and isopentenyl diphosphate (IPP) isomerase activities. GPP and FPP synthase formed GPP and E,E-FPP, respectively, as the sole products of the enzymatic condensation of IPP and dimethylallyl diphosphate (DMAPP). The properties of both enzymes are broadly similar to those of other prenyltransferases. The apparent native molecular masses are 54 +/- 3 kDa for GPP synthase and 110 +/- 6 kDa fo     

A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity

Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg²⁺ ions lead to the production of FPP, while the presence of Co²⁺ ions lead to geranyl diphosphate (GPP) production. In the presence of Mg²⁺ the AaFPPS affinity for allylic substrates is GPP > DMAPP > IPP. These results suggest that AaFPPS displays “catalytic promiscuity”, changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways.     

Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta

Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.     

Crystal structures of ligand‐bound octaprenyl pyrophosphate synthase from Escherichia coli reveal the catalytic and chain‐length determining mechanisms

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C₄₀ long‐chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans‐prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo‐form as well as in complexes with IPP and a FPP thio‐analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active‐site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand‐bound crystal structures of a long‐chain trans‐prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37–45. © 2014 Wiley Periodicals, Inc.     

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.     

Farnesyl Diphosphate Synthase. A Paradigm for Understanding Structure and Function Relationships in E-polyprenyl Diphosphate Synthases

The chain elongation reaction catalyzed by polyprenyl diphosphate synthases is the fundamental building reaction in the isoprenoid pathway. During chain elongation, the hydrocarbon moiety in an allylic isoprenoid diphosphate is added to the carbon–carbon double bond of isopentenyl diphosphate (IPP). The chain elongation enzymes can be divided into two genetically different families depending on whether the stereochemistry of the newly formed double bond during each cycle of chain elongation is E or Z. Farnesyl diphosphate (FPP) synthase, a member of the E-double bond family, is the best studied of the chain elongation enzymes and serves as a paradigm for understanding the reactions catalyzed by E-polyprenyl diphosphate synthases. The mechanism for chain elongation is a stereoselective electrophilic alkylation of the carbon–carbon double bond in IPP by the allylic substrate. X-ray structures of avian and E. coli FPP synthases have provided important insights about the mechanism for chain elongation and a structural basis for understanding the stereochemistry of the reaction.This review is dedicated to Professor Rodney Croteau on the occasion of his 60th birthday.     

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. 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 alpha3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg(2+). The new structure provides a plausible mechanism for the catalysis of UPPs.     

A thermophilic cyanobacterium Synechococcus elongatus has three different Class I prenyltransferase genes

Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C₅) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C₂₀) diphosphate synthase, another encodes a farnesyl (C₁₅) diphosphate synthase whose optimal reaction temperature is 60 °C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 °C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C₂₅), hexaprenyl (C₃₀), and heptaprenyl (C₃₅) diphosphates from dimethylallyl (C₅) diphosphate, geranyl (C₂₀) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).     

Structure, mechanism and function of prenyltransferases.

In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis-type and trans-type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5-epi-aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.     

Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil,Anthonomus grandis

Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full‐length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three‐dimensional structure of coleopteran FPPS was determined and compared to the X‐ray crystal structure of avian FPPS. The α‐helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS. © 2009 Wiley Periodicals, Inc.     

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

Significance of highly conserved aromatic residues in Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase

Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al. (2001) J. Biol. Chem. 276, 28459-28464]. In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis. The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined. All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wild-type, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme. Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1,600-fold decreases in the kcat/Km values compared to that of the wild-type but the Km values for both substrates IPP and FPP resulted in only moderate changes. Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the Km value for IPP and a 1,000-2,000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km values as compared to those of the wild-type. These results suggested that these conserved aromatic residues play important roles in the binding with both substrates, IPP and FPP, as well as the catalytic function of undecaprenyl diphosphate synthase.     

Substrate specificities of farnesyl diphosphate synthases from Bacillus stearothermophilus and porcine liver with cyclic substrate homologs

We investigated the substrate specificity of farnesyl diphosphate (FPP) synthase derived from Bacillus stearothermophilus and porcine liver by examining the reactivities of two cyclic substrate homologs, cyclohexylideneethyl diphosphate and cyclohexenylethyl diphosphate.Reaction of geranyl diphosphate with 2-cyclohexenylethyl diphosphate using bacterial or porcine liver FPP synthase produced (S)-geranylcyclohexylideneethyl diphosphate, with relative yields of 13.6% for the bacterial enzyme and 42.2% for the porcine liver enzyme. Reaction of cyclohexylideneethyl diphosphate with isopentenyl diphosphate produced 10-cyclohexyliden-3,7-dimethyldeca-2,6-dien-1-ol as a double condensation product, with relative yields of 23.1% (bacterial enzyme) and 3.0% (porcine liver enzyme). Reaction of cyclohexylideneethyl diphosphate with 2-cyclohexenylethyl diphosphate using bacterial enzyme produced (cyclohexylideneethyl)-cyclohexylideneethyl diphosphate (0.8% yield).