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The peptidoglycan exoskeleton shapes bacteria and protects them against osmotic forces, making its synthesis the target of many current antibiotics. Peptidoglycan precursors are attached to a lipid carrier and flipped from the cytoplasm into the periplasm to be incorporated into the cell wall. In Escherichia coli , this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS). E. coli MG1655 exhibits wild-type morphology at all temperatures, but one of our laboratory strains (CS109) was highly aberrant when grown at 42°C. This strain contained mutations affecting the Und-P synthetic pathway genes uppS , ispH , and idi Normal morphology was restored by overexpressing uppS or by replacing the mutant ( uppS31 ) with the wild-type allele. Importantly, moving uppS31 into MG1655 was lethal even at 30°C, indicating that the altered enzyme was highly deleterious, but growth was restored by adding the CS109 versions of ispH and idi Purified UppS W31R was enzymatically defective at all temperatures, suggesting that it could not supply enough Und-P during rapid growth unless suppressor mutations were present. We conclude that cell wall synthesis is profoundly sensitive to changes in the pool of polyisoprenoids and that isoprenoid homeostasis exerts a particularly strong evolutionary pressure. IMPORTANCE Bacterial morphology is determined primarily by the overall structure of the semirigid macromolecule peptidoglycan. Not only does peptidoglycan contribute to cell shape, but it also protects cells against lysis caused by excess osmotic pressure. Because it is critical for bacterial survival, it is no surprise that many antibiotics target peptidoglycan biosynthesis. However, important gaps remain in our understanding about how this process is affected by peptidoglycan precursor availability. Here, we report that a mutation altering the enzyme that synthesizes Und-P prevents cells from growing at high temperatures and that compensatory mutations in enzymes functioning upstream of uppS can reverse this phenotype. The results highlight the importance of Und-P metabolism for maintaining normal cell wall synthesis and shape., (Copyright © 2018 American Society for Microbiology.)

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co(2+) or Mn(2+) as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg(2+). In reaction with Co(2+), PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg(2+), PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co(2+), Mn(2+), or Mg(2+) in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.

A group of prenyltransferases produce linear lipids by catalyzing consecutive condensation reactions of farnesyl diphosphate (FPP) with specific numbers of isopentenyl diphosphate (IPP), a common building block of isoprenoid compounds. Depending on the stereochemistry of the double bonds formed during IPP condensation, these prenyltransferases are categorized as cis- and trans-types. Undecaprenyl diphosphate synthase (UPPS) that catalyzes chain elongation of FPP by consecutive condensation reactions with eight IPP, to form C₅₅ lipid carrier for bacterial cell wall biosynthesis, serves as a model for understanding cis-prenyltransferases. In this review, the current knowledge in UPPS kinetics, mechanisms, structures, and inhibitors is summarized.

Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 A) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 A). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 alpha-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an alpha-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.

The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene (PaIDS 4) encodes a farnesyl diphosphate synthase and the other two (PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.

A short-chain prenyl diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for farnesyl diphosphate synthase, ispA, was separated from other prenyl diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate and geranylgeranyl diphosphate.

The chromosomal ispA gene encoding farnesyl diphosphate synthase of Escherichia coli was disrupted by inserting a neo gene cassette. The null ispA mutants were viable. The growth yield of the mutants was 70% to 80% of that of the wild-type strain under aerobic conditions, and was almost the same as the wild-type under anaerobic conditions. The levels of ubiquinone-8 and menaquinone-8 were both significantly lower (less than 13% and 18% of normal, respectively) in the mutants than in the wild-type. The undecaprenyl phosphate level in the mutants was modestly lower (40% to 70% of normal) than in the wild-type strain. Thus the synthesis of all-E-octaprenyl diphosphate, the precursor of ubiquinone-8 and menaquinone-8, was decreased more severely than that of Z,E-mixed undecaprenyl diphosphate, the precursor of undecaprenyl monophosphates, under the conditions where the synthesis of farnesyl diphosphate was decreased. The condensation of isopentenyl diphosphate with dimethylallyl diphosphate was detected in the cell-free extracts of the mutants, although it was 5% of that in the wild-type strain. A low level of farnesyl diphosphate seems to be synthesized in the mutants by other prenyltransferases such as octaprenyl diphosphate synthase or undecaprenyl diphosphate 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. We now show that the monophosphate analogue of FPP binds UPPS with an eight times lower affinity (K(d)=4.4 microM) compared with the pyrophosphate analogue, a result of a larger dissociation rate constant (k(off)=192 s(-1)). Farnesol (1 mM) lacking the pyrophosphate does not inhibit the UPPS reaction. GGPP (geranylgeranyl pyrophosphate) containing a larger C20 hydrocarbon tail is an equally good substrate (K(m)=0.3 microM and kcat=2.1 s(-1)) compared with FPP. The shorter C10 GPP (geranyl pyrophosphate) displays a 90-fold larger K(m) value (36.0+/-0.1 microM) but similar kcat value (1.7+/-0.1 s(-1)) compared with FPP. Replacement of L85, L88 or F89 with Ala increases FPP and GGPP K(m) values by the same amount, indicating that these amino acids are important for substrate binding, but do not determine substrate specificity. With GGPP as a substrate, UPPS still catalyses eight isopentenyl pyrophosphate condensation reactions to synthesize C60 product. Computer modelling suggests that the upper portion of the active-site tunnel, where cis double bonds of the product reside, may be critical for determining the final product chain length.

Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. Recently, it was demonstrated that open reading frame Rv2361c of the Mycobacterium tuberculosis H37Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem. 275:22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an Escherichia coli expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryl diphosphate, omega,E,E-farnesyl diphosphate, omega,E,Z-farnesyl diphosphate, or omega,E,E,E-geranylgeranyl diphosphate, with Km values for the allylic substrates of 490, 29, 84, 290, and 40 microM, respectively. The Km value for isopentenyl diphosphate is 89 microM. The catalytic efficiency is greatest when omega,E,Z-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from M. tuberculosis. This is the first report of a bacterial Z-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the alpha-isoprene unit in the Z configuration, indicating that Rv1086 (omega,E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis.

Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.

The genes for chrysanthemyl diphosphate (CPP) synthase and farnesyl diphosphate (FPP) synthase from sagebrush, Artemisia tridentata spiciformis, were used to prepare a series of chimeric proteins to investigate the 1'-4 chain elongation, 1'-2 branching, and c1'-2-3 cyclopropanation reactions that join isoprenoid units to build more complex structures. The two genes were modified by site-directed mutagenesis to generate an identical set of six unique restriction sites at identical locations. The locations were selected to place a restriction site between each of the five conserved regions found in prenyltransferases that catalyze chain elongation. A series of chimeric proteins were generated by replacing amino acids in FPP synthase, beginning at the N-terminus of the enzyme, with increasing stretches of peptide from CPP synthase. An analysis of the products produced by the chimeras revealed a transition from 1'-4 chain elongation, to 1'-2 branching, and ultimately to c1'-2-3 cyclopropanation. These results demonstrate that the catalytic site for chain elongation, with minor modifications in its architecture, also catalyzes 1'-2 branching and c1'-2-3 cyclopropanation, and suggest that the branching and cyclopropanation reactions, in analogy to chain elongation, are electrophilic alkylations.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all-trans C40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity. Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton [12]. In order to understand the mechanism of enzyme activation for E. coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s(-1)) was 100 times larger than the steady-state rate (0.02 s(-1)). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s(-1)) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.

We found in the Escherichia coli genome sequence a homologue of RER2, a Saccharomyces cerevisiae gene required for proper localization of an endoplasmic reticulum protein, and designated it rth (RER2 homologue). The disruption of this gene was lethal for E. coli. To reveal its biological function, we isolated temperature-sensitive mutants of the rth gene. The mutant cells became swollen and burst at the nonpermissive temperature, indicating that their cell wall integrity was defective. Further analysis showed that the mutant cells were deficient in the activity of cis-prenyltransferase, namely, undecaprenyl diphosphate synthase, a key enzyme of the carrier lipid formation of peptidoglycan synthesis. The cellular level of undecaprenyl phosphate was in fact markedly decreased in the mutants. These results are consistent with the fact that the Rer2 homologue of Micrococcus luteus shows undecaprenyl diphosphate synthase activity (N. Shimizu, T. Koyama, and K. Ogura, J. Biol. Chem. 273:19476-19481, 1998) and demonstrate that E. coli Rth is indeed responsible for the maintenance of cell wall rigidity. Our work on the yeast rer2 mutants shows that they are defective in the activity of cis-prenyltransferase, namely, dehydrodolichyl diphosphate synthase, a key enzyme of dolichol synthesis. Taking these data together, we conclude that the RER2 gene family encodes cis-prenyltransferase, which plays an essential role in cell wall biosynthesis in bacteria and in dolichol synthesis in eukaryotic cells and has been well conserved during evolution.

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with (E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.

Different organisms produce different species of isoprenoid quinones, each with its own distinctive length. These differences in length are commonly exploited in microbial classification. The side chain length of quinone is determined by the nature of the polyprenyl diphosphate synthase that catalyzes the reaction. To determine if the side chain length of ubiquinone (UQ) has any distinct role to play in the metabolism of the cells in which it is found, we cloned the solanesyl diphosphate synthase gene (sdsA) from Rhodobacter capsulatus SB1003 and expressed it in Escherichia coli and Saccharomyces cerevisiae. Sequence analysis revealed that the sdsA gene encodes a 325-amino-acid protein which has similarity (27 to 40%) with other prenyl diphosphate synthases. Expression of the sdsA gene complemented a defect in the octaprenyl diphosphate synthase gene of E. coli and the nonrespiratory phenotype resulting from a defect in the hexaprenyl diphosphate synthase gene of S. cerevisiae. Both E. coli and S. cerevisiae expressing the sdsA gene mainly produced solanesyl diphosphate, which resulted in the synthesis of UQ-9 without any noticeable effect on the growth of the cells. Thus, it appears that UQ-9 can replace the function of UQ-8 in E. coli and UQ-6 in S. cerevisiae. Taken together with previous results, the results described here imply that the side chain length of UQ is not a critical factor for the survival of microorganisms.

Farnesol strongly inhibited growth of a halophilic archaeon, Haloferax volcanii, with an IC50 value of only 2 microM (0.4 microgram/ml) in rich medium and 50 nM (0.01 microgram/ml) in minimal medium without lysis. Other isoprenoid alcohols such as isopentenol, dimethylallyl alcohol, geraniol, and geranylgeraniol at 500 microM did not affect its growth. Mevalonate, which is the precursor of all isoprenoid membrane lipids in archaea, led to recovery of the growth inhibition of H. volcanii, but acetate had no such effect. Farnesol inhibited incorporation of acetate, but not mevalonate, into the lipid fraction. These results suggest that farnesol inhibited the biosynthetic pathway from acetate (acetyl-CoA) to mevalonate. Farnesol is known to be derived from the important intermediate of isoprenoids, farnesyl diphosphate (FPP), and found in neutral lipid fraction from this archaeon. Moreover, the cell-free extracts from H. volcanii could phosphorylate farnesol with ATP to generate farnesyl monophosphate and FPP. We conclude that farnesol-mediated isoprenoid synthesis regulation system by controlling farnesol concentration is present in H. volcanii.

A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39,689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase.

Rat liver microsomes catalyzed the formation of A,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate in the presence of Triton X-100. Studies on product specificity using various primers such as Z,E-farnesyl diphosphate, E,E-farnesyl diphosphate, Z,E,E-geranylgeranyl diphosphate, E,E,E-geranylgeranyl diphosphate, Z,E,E,E-geranylfarnesyl diphosphate, and E,E,E,E-geranylfarnesyl diphosphate suggested that the microsomal dehydrodolichyl diphosphate synthase has such properties that it releases Z,E,E-geranylgeranyl diphosphate, the first intermediate, in the reactions with farnesyl diphosphate as the starting primer. Metabolic labeling of rat liver slices with [2-3H]mevalonic acid revealed the accumulation of E,E,E-geranylgeranyl (di)phosphates as well as dolichyl (di)phosphate (C85 and C90) and dehydrodolichol (C85 and C90), but no accumulation of Z,E,E-geranylgeranyl (di)phosphate or E,E-farnesyl (di)phosphate was detected. Microsomal enzyme preparations from mouse liver and hamster liver also produced Z,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate.

Isopentenyl pyrophosphate (IPP) isomerase, farnesyl pyrophosphate (FPP) synthetase, octaprenyl pyrophosphate (OPP) synthetase and undecaprenyl pyrophosphate (UPP) synthetase were partially purified from Escherichia coli by DEAE-Toyopearl chromatography. FPP synthetase catalyzed the condensation of IPP with dimethylallyl pyrophosphate (DPP) as well as with geranyl pyrophosphate (GPP) to yield FPP as final product. OPP synthetase and UPP synthetase catalyzed the condensation of IPP with FPP to yield OPP and cis,trans-polyprenyl pyrophosphates (the C45-, C50, and C55-compound), respectively. Neither DPP nor GPP acted as a priming substrate for either enzyme. These four enzymes required Mg2+ or Mn2+ for their activities. UPP synthetase required also Triton X-100 for its activity. The addition of Triton X-100 enhanced OPP synthetase, but it did not affect IPP isomerase and FPP synthetase. It seems possible that the combination of the four enzymes ensures the in vivo synthesis of long-chain isoprenoids in E. coli.

Geranyl pyrophosphate synthetase, which catalyzes the condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate to give geranyl pyrophosphate, was purified 490-fold from Micrococcus luteus extracts by DEAE-Sephadex, hydroxylapatite, and Sephadex G-100 column chromatography. The enzyme has a pH optimum at 7.7 and the molecular weight was estimated to be 70,000 by Sephadex gel filtration. The Km values for isopentenyl pyrophosphate and dimethylallyl pyrophosphate were 8 microM and 62 microM, respectively. The enzyme required Mg2+ for maximum activity. Tween 80 showed a stimulative effect whereas Triton X-100 was rather inhibitory on the enzyme activity. Inorganic pyrophosphate and iodoacetamide were both potent inhibitors of the enzyme. The purified enzyme fraction was also capable of catalyzing the synthesis of geranylgeranyl pyrophosphate from isopentenyl pyrophosphate and farnesyl pyrophosphate, but lacked geranyl-transferring activity. The catalytic activities of geranylgeranyl pyrophosphate synthesis and geranyl pyrophosphate synthesis were affected differently by iodoacetamide and Triton X-100. This enzyme fraction may be a mixture of two enzymes, geranyl pyrophosphate synthetase and geranylgeranyl pyrophosphate synthetase catalyzing the reactions of C5 greater than C10 and C15 greater than C20, respectively, or a single enzyme with two independent catalytic sites responsible for the C5 greater than C10 and C15 greater than C20 reactions. In any case, the existence of a new geranylgeranyl pyrophosphate synthetase different from the known geranylgeranyl pyrophosphate synthetase catalyzing the continuous condensation reaction of C5 greater than C10 greater than C15 greater than C20 was demonstrated.