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1.  Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase 
Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components.
Putative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)- over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R)-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix α2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding.
Our new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.
PMCID: PMC1851959  PMID: 17397541
2.  Discovery of a metabolic alternative to the classical mevalonate pathway 
eLife  2013;2:e00672.
Eukarya, Archaea, and some Bacteria encode all or part of the essential mevalonate (MVA) metabolic pathway clinically modulated using statins. Curiously, two components of the MVA pathway are often absent from archaeal genomes. The search for these missing elements led to the discovery of isopentenyl phosphate kinase (IPK), one of two activities necessary to furnish the universal five-carbon isoprenoid building block, isopentenyl diphosphate (IPP). Unexpectedly, we now report functional IPKs also exist in Bacteria and Eukarya. Furthermore, amongst a subset of species within the bacterial phylum Chloroflexi, we identified a new enzyme catalyzing the missing decarboxylative step of the putative alternative MVA pathway. These results demonstrate, for the first time, a functioning alternative MVA pathway. Key to this pathway is the catalytic actions of a newly uncovered enzyme, mevalonate phosphate decarboxylase (MPD) and IPK. Together, these two discoveries suggest that unforeseen variation in isoprenoid metabolism may be widespread in nature.
eLife digest
Living things make thousands of chemicals that are vital for life, and are also useful as medicines, perfumes, and food additives. The largest family of these natural chemicals is called the isoprenoids, and members of this family are found in all three domains of life: the eukaryotes (such as plants and animals), the Archaea (an ancient group of single-celled microbes), and bacteria.
The isoprenoids are made from a smaller building block called isopentenyl diphosphate, IPP for short, that contains five carbon atoms and two phosphate groups. IPP can be produced in two ways. The classical mevalonate pathway is found in most eukaryotes, including humans; statin drugs are used to inhibit this pathway to treat those with high cholesterol and reduce the risk of heart disease. The second pathway does not use the compound mevalonate and is found in many, but not all, bacteria as well as the chloroplasts of plants. Until recently, however, the enzymes needed for the last two steps of the classical mevalonate pathway appeared to be missing in the Archaea and in some bacteria.
Researchers subsequently discovered that an enzyme called isopentenyl phosphate kinase, shortened to IPK, was responsible for one of these two missing steps—the addition of IPP’s second phosphate group. The way this enzyme worked also suggested that there was an alternative mevalonate pathway in which the order of the last two steps was reversed. However, the identity of the enzyme responsible for the other step—the removal of a molecule of carbon dioxide to make the starting material needed by IPK—remained mysterious.
Now Dellas et al. have discovered the enzyme responsible for this missing step in Green non-sulphur bacteria, confirming the existence of the alternative mevalonate pathway for the first time. Previously it had been thought that this enzyme acted in the classical mevalonate pathway; but in fact this enzyme has evolved a new function and is not involved in the classical pathway at all. Moreover, Dellas et al. show that Green non-sulphur bacteria, and some eukaryotes, also have functional IPK enzymes. This means that IPK has now unexpectedly been observed in all three domains of life, and hints at another target to medically control mevalonate pathways. The discovery of the missing enzyme in the alternative pathway opens the door to the re-examination of many other living things, to find which have the new pathway and to work out why.
PMCID: PMC3857490  PMID: 24327557
Mevalonate pathway; Isopentenyl diphosphate; Archaea; Mevalonate phosphate decarboxylase; Chloroflexi; Plants; Arabidopsis; Other
3.  Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway 
Journal of Bacteriology  2014;196(5):1055-1063.
Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.
PMCID: PMC3957691  PMID: 24375100
4.  Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase† 
Biochemistry  2012;51(28):5611-5621.
Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg++-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g. Staphylococcus, Streptococcus and Enterococcus spp.) and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of small molecule (i.e. metabolite) kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo-, MVAPP-bound and ternary complexed wild-type MDD provides structural information on the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (decreased kcat of 103-fold and 105-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp283 functions as a catalytic base and is essential to the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop (‘P-loop’) provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.
PMCID: PMC4227304  PMID: 22734632
5.  Identification, Evolution, and Essentiality of the Mevalonate Pathway for Isopentenyl Diphosphate Biosynthesis in Gram-Positive Cocci 
Journal of Bacteriology  2000;182(15):4319-4327.
The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)–pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.
PMCID: PMC101949  PMID: 10894743
6.  The Sorbitol Phosphotransferase System Is Responsible for Transport of 2-C-Methyl-d-Erythritol into Salmonella enterica Serovar Typhimurium 
Journal of Bacteriology  2004;186(2):473-480.
2-C-methyl-d-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-d-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-d-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-d-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-d-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-d-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-d-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-d-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-d-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.
PMCID: PMC305747  PMID: 14702317
7.  A triclinic crystal form of Escherichia coli 4-diphosphocytidyl-2C-methyl-d-erythritol kinase and reassessment of the quaternary structure 
The structure of a triclinic crystal form of 4-diphosphocytidyl-2C-methyl-d-erythritol kinase has been determined. Comparisons with a previously reported monoclinic crystal form raise questions about our knowledge of the quaternary structure of this enzyme.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE; EC contributes to the 1-deoxy-d-xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and Gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-­diphosphos­phocytidyl-2C-methyl-d-erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C-methyl-d-erythritol. A triclinic crystal structure of the Escherichia coli IspE–ADP complex with two molecules in the asymmetric unit was determined at 2 Å resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the ‘triclinic dimer’ is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization.
PMCID: PMC2833027  PMID: 20208151
mevalonate-independent pathway; isoprenoid biosynthesis; kinases
8.  Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway 
Journal of Bacteriology  2004;186(1):61-67.
It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent Km values for R,S-mevalonate and ATP were 41 and 339 μM, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the Ki values were 46 and 45 μM for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (Ki difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.
PMCID: PMC303434  PMID: 14679225
9.  Evidence of a Novel Mevalonate Pathway in Archaea 
Biochemistry  2014;53(25):4161-4168.
Isoprenoids make up a remarkably diverse class of more than 25000 biomolecules that include familiar compounds such as cholesterol, chlorophyll, vitamin A, ubiquinone, and natural rubber. The two essential building blocks of all isoprenoids, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are ubiquitous in the three domains of life. In most eukaryotes and archaea, IPP and DMAPP are generated through the mevalonate pathway. We have identified two novel enzymes, mevalonate-3-kinase and mevalonate-3-phosphate-5-kinase from Thermoplasma acidophilum, which act sequentially in a putative alternate mevalonate pathway. We propose that a yet unidentified ATP-independent decarboxylase acts upon mevalonate 3,5-bisphosphate, yielding isopentenyl phosphate, which is subsequently phosphorylated by the known isopentenyl phosphate kinase from T. acidophilum to generate the universal isoprenoid precursor, IPP.
PMCID: PMC4081127  PMID: 24914732
10.  The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene 
Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway.
PMCID: PMC4357925  PMID: 25636853
11.  Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis†  
Journal of Bacteriology  2002;184(8):2116-2122.
Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, ΔHa and Km values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar Ki for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.
PMCID: PMC134966  PMID: 11914342
12.  Subcellular evidence for the involvement of peroxisomes in plant isoprenoid biosynthesis 
Plant Signaling & Behavior  2011;6(12):2044-2046.
The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al.1 revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP.2 This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle.3 Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells.
PMCID: PMC3337203  PMID: 22080790
5-phosphomevalonate kinase; Arabidopsis thaliana; Catharanthus roseus; farnesyl diphosphate synthase; isoprenoid; mevalonate 5-diphosphate decarboxylase; mevalonic acid pathway; peroxisome
13.  Gγ1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration 
PLoS Genetics  2009;5(1):e1000333.
The isoprenoid biosynthetic pathway leading from the production of mevalonate by HMGCoA reductase (Hmgcr) to the geranylation of the G protein subunit, Gγ1, plays an important role in cardiac development in the fly. Hmgcr has also been implicated in the release of the signaling molecule Hedgehog (Hh) from hh expressing cells and in the production of an attractant that directs primordial germ cells to migrate to the somatic gonadal precursor cells (SGPs). The studies reported here indicate that this same hmgcr→Gγ1 pathway provides a novel post-translational mechanism for modulating the range and activity of the Hh signal produced by hh expressing cells. We show that, like hmgcr, gγ1 and quemao (which encodes the enzyme, geranylgeranyl diphosphate synthetase, that produces the substrate for geranylation of Gγ1) are components of the hh signaling pathway and are required for the efficient release of the Hh ligand from hh expressing cells. We also show that the hmgcr→Gγ1 pathway is linked to production of the germ cell attractant by the SGPs through its ability to enhance the potency of the Hh signal. We show that germ cell migration is disrupted by the loss or gain of gγ1 activity, by trans-heterozygous combinations between gγ1 and either hmgcr or hh mutations, and by ectopic expression of dominant negative Gγ1 proteins that cannot be geranylated.
Author Summary
Previous studies have shown that HMGCoA reductase (Hmgcr) is required for the production of a germ cell attractant by the somatic gonadal precursor cells (SGPs) and for the release of the Hedgehog (Hh) ligand by hh expressing cells. However, it was not clear what role mevalonate, the biosynthetic product of Hmgcr, played in either of these processes or whether the hmgcr-dependent germ cell attractant corresponds to the Hh ligand (which is known to be expressed by the SGPs). We show here that the downstream target for Hmgcr both in generating the germ cell attractant and in releasing the Hh ligand is the G protein, Gγ1. Gγ1 must be geranylated in order to function, and the substrate for this posttranslational modification, geranylgeranyl-pyrophosphate, is one of the biosynthetic products of mevalonate. In addition to demonstrating a critical role for Gγ1 (as well as the hmgcr isoprenoid biosynthetic pathway) in releasing Hh from hh expressing cells, our findings provide additional evidence that Hh protein produced by the SGPs is an hmgcr-dependent germ cell attractant.
PMCID: PMC2607556  PMID: 19132091
14.  Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in Methanothermobacter thermautotrophicus 
Journal of Bacteriology  2004;186(6):1811-1817.
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-d-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70°C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: Km, 64 μM; specific activity, 0.476 μmol mg−1 min−1; and kcat, 1.6 s−1.
PMCID: PMC355898  PMID: 14996812
15.  Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis†  
Journal of Bacteriology  2002;184(15):4065-4070.
Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni2+-agarose to apparent homogeneity and a specific activity of 10 μmol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s20,w, 5.3). Optimal activity occurred in 2.0 mM MgCl2 at 37oC. The ΔHa was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pKa of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 ± 0.2 and that of covalent acetylation was 0.60 ± 0.02. The Km for the hydrolysis of acetyl-CoA was 10 μM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.
PMCID: PMC135212  PMID: 12107122
16.  Functional Evaluation of Conserved Basic Residues in Human Phosphomevalonate Kinase.† 
Biochemistry  2007;46(42):11780-11788.
Phosphomevalonate kinase (PMK) catalyzes the cation dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. Based on this precedent, conserved basic residues of human PMK have been mutated and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished Vmax values in both reaction directions (>1000-fold) with only slight Km perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated Km values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60 and 30-fold, respectively) as well as decreases (50-fold (forward) and 85-fold (reverse)) in Vmax. R84M also exhibits inflated Km values (50 and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The Ki values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45- and 63-fold; effects are comparable to the 30- and 38-fold inflations in Km for mevalonate 5-diphosphate. R141M exhibits little perturbation in Vmax (14-fold (forward) and 10-fold (reverse)) but has inflated Km values for ATP and ADP (48 and 136-fold, respectively). The Kd of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.
PMCID: PMC2530820  PMID: 17902708
17.  The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity. 
Journal of Bacteriology  1997;179(15):4664-4670.
The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids. We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C. Chambon, V. Ladeveve, M. Servouse, L. Blanchard, C. Javelot, B. Vladescu, and F. Karst, Lipids 26:633-636, 1991) to characterize the mutated allele. We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant. Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted. Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure. Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation.
PMCID: PMC179309  PMID: 9244250
18.  Conformational Dynamics of the Flexible Catalytic Loop in Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase 
Chemical biology & drug design  2009;73(1):26-38.
In mycobacteria, the biosynthesis of the precursors to the essential isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate is carried out by the methylerythritol phosphate (MEP) pathway. This route of synthesis is absent in humans, who utilize the alternative mevalonate acid (MVA) route, thus making the enzymes of the MEP pathway of chemotherapeutic interest. One such identified target is the second enzyme of the pathway, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). Only limited information is currently available concerning the catalytic mechanism and structural dynamics of this enzyme, and only recently has a crystal structure of Mycobacterium tuberculosis species of this enzyme been resolved including all factors required for binding. Here, the dynamics of the enzyme is studied in complex with NADPH, Mn2+, in the presence and absence of the fosmidomycin inhibitor using conventional molecular dynamics and an enhanced sampling technique, Reversible Digitally Filtered Molecular Dynamics. The simulations reveal significant differences in the conformational dynamics of the vital catalytic loop between the inhibitor-free and inhibitor-bound enzyme complexes and highlight the contributions of conserved residues in this region. The substantial fluctuations observed suggest that DXR may be a promising target for computer-aided drug discovery through the relaxed complex method.
PMCID: PMC2982673  PMID: 19152632
The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge.
PMCID: PMC3026612  PMID: 20932952
mevalonate pathway; isoprenoid biosynthesis; HMG-CoA; sterol biosynthesis
20.  Transcriptome exploration of the sex pheromone gland of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) 
Parasites & Vectors  2013;6:56.
Molecules involved in pheromone biosynthesis may represent alternative targets for insect population control. This may be particularly useful in managing the reproduction of Lutzomyia longipalpis, the main vector of the protozoan parasite Leishmania infantum in Latin America. Besides the chemical identity of the major components of the L. longipalpis sex pheromone, there is no information regarding the molecular biology behind its production. To understand this process, obtaining information on which genes are expressed in the pheromone gland is essential.
In this study we used a transcriptomic approach to explore the pheromone gland and adjacent abdominal tergites in order to obtain substantial general sequence information. We used a laboratory-reared L. longipalpis (one spot, 9-Methyl GermacreneB) population, captured in Lapinha Cave, state of Minas Gerais, Brazil for this analysis.
From a total of 3,547 cDNA clones, 2,502 high quality sequences from the pheromone gland and adjacent tissues were obtained and assembled into 1,387 contigs. Through blast searches of public databases, a group of transcripts encoding proteins potentially involved in the production of terpenoid precursors were identified in the 4th abdominal tergite, the segment containing the pheromone gland. Among them, protein-coding transcripts for four enzymes of the mevalonate pathway such as 3-hydroxyl-3-methyl glutaryl CoA reductase, phosphomevalonate kinase, diphosphomevalonate descarboxylase, and isopentenyl pyrophosphate isomerase were identified. Moreover, transcripts coding for farnesyl diphosphate synthase and NADP+ dependent farnesol dehydrogenase were also found in the same tergite. Additionally, genes potentially involved in pheromone transportation were identified from the three abdominal tergites analyzed.
This study constitutes the first transcriptomic analysis exploring the repertoire of genes expressed in the tissue containing the L. longipalpis pheromone gland as well as the flanking tissues. Using a comparative approach, a set of molecules potentially present in the mevalonate pathway emerge as interesting subjects for further study regarding their association to pheromone biosynthesis. The sequences presented here may be used as a reference set for future research on pheromone production or other characteristics of pheromone communication in this insect. Moreover, some matches for transcripts of unknown function may provide fertile ground of an in-depth study of pheromone-gland specific molecules.
PMCID: PMC3632494  PMID: 23497448
Lutzomyia longipalpis; Male pheromone gland; Transcriptome; Mevalonate pathway
21.  A Gene Cluster for the Mevalonate Pathway from Streptomyces sp. Strain CL190 
Journal of Bacteriology  2000;182(15):4153-4157.
A biosynthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC, the rate-limiting enzyme of the mevalonate pathway for isopentenyl diphosphate biosynthesis, had previously been purified from Streptomyces sp. strain CL190 and its corresponding gene (hmgr) had been cloned (S. Takahashi, T. Kuzuyama, and H. Seto, J. Bacteriol. 181:1256–1263, 1999). Sequence analysis of the flanking regions of the hmgr gene revealed five new open reading frames, orfA to -E, which showed similarity to those encoding eucaryotic and archaebacterial enzymes for the mevalonate pathway. Feeding experiments with [1-13C]acetate demonstrated that Escherichia coli JM109 harboring the hmgr gene and these open reading frames used the mevalonate pathway under induction with isopropyl β-d-thiogalactopyranoside. This transformant could grow in the presence of fosmidomycin, a potent and specific inhibitor of the nonmevalonate pathway, indicating that the mevalonate pathway, intrinsically absent in E. coli, is operating in the E. coli transformant. The hmgr gene and orfABCDE are thus unambiguously shown to be responsible for the mevalonate pathway and to form a gene cluster in the genome of Streptomyces sp. strain CL190.
PMCID: PMC101890  PMID: 10894721
22.  Development of a multi-gene expression system in Xanthophyllomyces dendrorhous 
Red yeast, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) that is widely used in the aquaculture, food, pharmaceutical and cosmetic industries. Recently, the potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Addition of mevalonate, the common precursor for isoprenoid biosynthesis, has been shown to be critical to enhance the astaxanthin content in X. dendrorhous. However, addition of mevalonate is unrealistic during industrial isoprenoid production because it is an unstable and costly chemical. Therefore, up-regulating the intracellular mevalonate supply by enhancing the mevalonate synthetic pathway though genetic engineering is a promising strategy to improve isoprenoid production in X. dendrorhous. However, a system to strongly express multiple genes has been poorly developed for X. dendrorhous.
Here, we developed a multiple gene expression system using plasmids containing three strong promoters in X. dendrorhous (actin, alcohol dehydrogenase and triose-phosphate isomerase) and their terminators. Using this system, three mevalonate synthetic pathway genes encoding acetoacetyl-CoA thiolase, HMG-CoA synthase and HMG-CoA reductase were overexpressed at the same time. This triple overexpressing strain showed an increase in astaxanthin production compared with each single overexpressing strain. Additionally, this triple overexpression of mevalonate synthetic pathway genes together with genes involved in β-carotene and astaxanthin synthesis showed a synergetic effect on increasing astaxanthin production. Finally, astaxanthin production was enhanced by 2.1-fold compared with the parental strain without a reduction of cell growth.
We developed a system to strongly overexpress multiple genes in X. dendrorhous. Using this system, the synthetic pathway of mevalonate, a common substrate for isoprenoid biosynthesis, was enhanced, causing an increase in astaxanthin production. Combining this multiple gene overexpression system with a platform strain that overproduces mevalonate has the potential to improve industrial production of various isoprenoids in X. dendrorhous.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0175-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4264253  PMID: 25471659
Isoprenoid; Carotenoid; Astaxanthin; Mevalonate; Xanthophyllomyces dendrorhous; Phaffia rhodozyma; Metabolic engineering; Cell factory
23.  The Mevalonate Pathway of Staphylococcus aureus▿ †  
Journal of Bacteriology  2008;191(3):851-861.
Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences.
PMCID: PMC2632080  PMID: 19028897
24.  Escherichia coli Open Reading Frame 696 Is idi, a Nonessential Gene Encoding Isopentenyl Diphosphate Isomerase 
Journal of Bacteriology  1999;181(15):4499-4504.
Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3′-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44°C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the Vmax of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in Kms. The E. coli protein requires Mg2+ or Mn2+ for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.
PMCID: PMC103578  PMID: 10419945
25.  Mevalonate Analogues as Substrates of Enzymes in the Isoprenoid Biosynthetic Pathway of Streptococcus pneumoniae 
Bioorganic & medicinal chemistry  2009;18(3):1124-1134.
Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway, which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this pathway appears to be regulated at the mevalonate kinase (MK) step, which is strongly feedback-inhibited by diphosphomevalonate (DPM), the penultimate compound in the pathway. The human mevalonate pathway is not regulated by DPM, making the bacterial pathway an attractive antibiotic target. Since DPM has poor drug characteristics, being highly charged, we propose to use unphosphorylated, cell-permeable prodrugs based on mevalonate that will be phosphorylated in turn by MK and phosphomevalonate kinase (PMK) to generate the active compound in situ. To test the limits of this approach, we synthesized a series of C3-substituted mevalonate analogues to probe the steric and electronic requirements of the MK and PMK active sites. MK and PMK accepted substrates with up to two additional carbons, showing a preference for small substitutents. This result establishes the feasibility of using a prodrug strategy for DPM-based antibiotics in S. pneumoniae and identified several analogues to be tested as inhibitors of MK. Among the substrates accepted by both enzymes were cyclopropyl, vinyl, and ethynyl mevalonate analogues that, when diphosphorylated, might be mechanism-based inactivators of the next enzyme in the pathway, diphosphomevalonate decarboxylase.
PMCID: PMC2842986  PMID: 20056424
Isoprenoid pathway; mevalonic acid; phosphomevalonic acid; diphosphomevalonic acid; mevalonate kinase; phosphomevalonate kinase; diphosphomevalonate decarboxylase; prodrug

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