Author response: Discovery of a metabolic alternative to the classical mevalonate pathway (2013)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract 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. https://doi.org/10.7554/eLife.00672.001 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. https://doi.org/10.7554/eLife.00672.002 Introduction Isoprenoids constitute a substantial family of primary and secondary metabolites in all three domains of life. These molecules play essential and specialized roles for their hosts including modulation of membrane fluidity (cholesterol, hopanoids, squalene), chemical defense and communication (mono-, sesqui- and diterpenes), photoprotection and energy transfer (carotenoids) (Lu and Li, 2008), and growth regulation (giberellins) (Hedden and Kamiya, 1997). Additionally, isoprenoids such as quinones, chlorophyll, bacteriochlorophyll, and some cellular proteins are tethered to a polyisoprenoid chain to direct localization to membranes or to potentiate interactions with other biomolecules (Nowicka and Kruk, 2010; Schafer and Rine, 1992). Isopentenyl diphosphate (IPP, 1) and its isomer, dimethylallyl diphosphate (DMAPP, 2), are the five-carbon building blocks of all higher order isoprenoids. It is currently accepted that IPP is biosynthesized via one of two metabolic pathways: the mevalonate (MVA or sometimes MEV) pathway (Katsuki and Bloch, 1967; Lynen, 1967) or the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway (also known as the 2-C-methyl-D-erythritol 4-phosphate, MEP, or Rohmer pathway) (Arigoni et al., 1997; Eisenreich et al., 1998; Rohmer, 1999). These two metabolic systems utilize non-homologous enzymes that evolved independently to produce the same 5-carbon end product, IPP (1). Typically, a given organism uses one pathway or the other (Lange et al., 2000). Eukarya appear to encode the classical MVA pathway (with some exceptions, see [Cassera et al., 2004]); plants additionally encode the DXP pathway that operates in the chloroplast. Most bacteria employ the DXP pathway with the exception of several phyla that contain full or partial MVA pathway genes (Bochar et al., 1999, Lombard and Moreira, 2011). While the domain Archaea and the bacterial class Chloroflexi do not encode gene homologs for the DXP pathway, nearly all of these species characterized genomically to date encode an incomplete classical MVA pathway. That is, many of these species lack identifiable genes encoding enzymes for one or both of the terminal steps of IPP biosynthesis through mevalonate. This observation is puzzling due to the essentiality of isoprenoid compounds in all organisms. Recent phylogenetic and experimental data suggest that Archaea (and probably also the Chloroflexi) encode an alternative MVA pathway, which bifurcates from the classical pathway following the phosphorylation of mevalonate to phosphomevalonate (MVAP, also known as mevalonate 5-phosphate, 3) (Grochowski et al., 2006; Matsumi et al., 2010; Miziorko, 2010) (Figure 1). Following MVAP biosynthesis, the classical MVA pathway uses phosphomevalonate kinase (PMK) to phosphorylate MVAP (3) producing diphosphomevalonate (MVAPP, also known as mevalonate 5-diphosphate, 5). MVAPP then undergoes a phosphorylation-dependent decarboxylation catalyzed by mevalonate 5-diphosphate decarboxylase (MDD, also known as MDC or DPM-DC) producing the key five-carbon isoprenoid building block, IPP (1). Figure 1 Download asset Open asset The classical and alternative MVA pathways. Both branches of the MVA pathway begin with acetyl-CoA (and acetoacetyl-CoA) and proceed through a series of enzymatic reactions involving 3-hydroxy-3-methylglutary-CoA Synthase (HMGS), 3-hydroxy-3-methylglutary-CoA Reductase (HMGR, the presumed early rate-limiting step), and mevalonate kinase (MVK) before branching. At the bifurcation, the canonical MVA pathway, highlighted by light blue arrows, guides MVAP (3) through an additional phosphorylation reaction followed by a phosphorylation-dependent decarboxylation carried out by phosphomevalonate kinase (PMK) and diphosphomevalonate decarboxylase (MDD), respectively. The alternative MVA pathway, highlighted with light brown arrows, hypothetically decarboxylates MVAP (3) prior to the phosphorylation reaction carried out by IPK but the former step has not been discovered until now (Grochowski et al., 2006). The enzymes MVK, PMK, MDD, and IPK all consume ATP during catalysis. All enzymes are shown in green type. Statins serve as inhibitors of HMGR as highlighted. https://doi.org/10.7554/eLife.00672.003 The alternative MVA pathway is posited to reverse these steps; decarboxylation of MVAP (3) followed by phosphorylation of isopentenyl phosphate (IP, 4) (Figure 1). The first step would require a currently undetected enzyme activity, MVAP decarboxylase (MPD). The final phosphorylation activity has been characterized in Archaea and is catalyzed by isopentenyl phosphate kinase (IPK), an ATP-dependent kinase that phosphorylates IP (4) forming IPP (1) (Figure 1). Until now, the IPK gene has not been studied among the Chloroflexi bacteria or within eukaryotic organisms presumably encoding all the enzymes of the classical MVA pathway. We performed extensive phylogenetic analyses to reveal a spotty distribution of IPK-bearing organisms in animals, several fungi, all plants, and some bacteria (including those from the class Chloroflexi). Following heterologous expression, purification, and kinetic characterization of several proteins encoding representative IPK-like genes from each domain of life, we show that fully active and highly specific IPKs unexpectedly exist outside of Archaea. One such fully active IPK was characterized from the Chloroflexi bacteria, Roseiflexus castenholzii. Interestingly, by classical genome annotation, the Chloroflexi bacteria, as well as certain other archaeal and bacterial groups, contain only a subset of components of the classical (PMK-, MDD-utilizing) and alternative (MPD-, IPK-utilizing) MVA pathways, resulting in what appears to be the existence of two incomplete branches of the MVA pathway in one organism (Figure 1). For example, Chloroflexi bacteria appear to be missing the PMK gene from the classical pathway and the MPD gene from the alternative pathway (Lombard and Moreira, 2011). This arrangement contrasts with archaeal species, which do not appear to encode either PMK or MDD from the classical MVA pathway. Through biochemical characterization of the enzymes encoded by the remaining MVA pathway genes (MDD and IPK) from the Chloroflexi bacterium, R. castenholzii, we unequivocally demonstrate that a functional alternative MVA pathway does exist and is encoded in a completely unexpected manner. In R. castenholzii, the putative MDD of the classical MVA pathway surprisingly is fully functional, and until now, expresses an unknown MPD activity associated with the alternative MVA pathway. Combined with our observation of a fully functional IPK-like gene from this same organism, these results provide the first definitive bioinformatic and experimental evidence for a fully operative alternative MVA pathway in nature. Results Phylogenetic analyses of IPK homologs IPK is a member of the amino acid kinase (AAK) superfamily. To date, the search for eukaryotic IPK-like genes has been limited since putative eukaryotic IPK homologs are divergent in sequence and can be difficult to distinguish from other AAK superfamily genes (Lombard and Moreira, 2011). Within IPK, we previously identified a catalytically essential active site histidine residue that is not conserved among AAK family members belonging to the amino acid kinase superfamily (Dellas and Noel, 2010). Using this structurally and functionally defined residue as an indication of possible IPK activity (Dellas and Noel, 2010), we next identified putative IPK homologs from all three domains of life. We used PSI-BLAST and profile Hidden Markov Models (HMMs) to detect IPK homologs in public protein, expressed sequence tag (EST), and genome databases. IPK homologs bearing the key histidine signature appear in nearly all archaea (Lombard and Moreira, 2011, Matsumi et al., 2010), a cluster of Chloroflexi bacteria (Lombard and Moreira, 2011), every sequenced green plant genome, and, in an exceptionally sporadic distribution, across most major eukaryotic lineages (Figure 2, Table 1). Figure 2 Download asset Open asset Phylogenetic distribution of IPK across the three domains of life. Maximum likelihood tree of selected IPK protein sequences. Eukaryotes are highlighted with blues, selected archaeal clades with grays and a small group of bacteria with purple. The tree is anchored by several bacterial fosfomycin kinases. See Figure 2—source data 1 for an alignment of IPK homologs. See Figure 2—source data 2 for a table of IPK homolog sequences. https://doi.org/10.7554/eLife.00672.004 Figure 2—source data 1 Alignment of IPKs from the three domains of life. https://doi.org/10.7554/eLife.00672.005 Download elife-00672-fig2-data1-v1.rtf Figure 2—source data 2 Additional IPK sequences from an assortment of sequence databases. https://doi.org/10.7554/eLife.00672.006 Download elife-00672-fig2-data2-v1.xlsx Table 1 Phylogenetic distribution of IPK in Eukarya https://doi.org/10.7554/eLife.00672.007 ClassificationIPK-bearing speciesSpecies with no IPKMammals35 mammalian genomesBirdsGallus gallusTaeniopygia guttataMeleagris gallopavoReptilesAnolis carolensisPhilodryas olfersii (EST)*AmphibiansNotophthalmus viridescens (EST)Xenopus tropicalisFishCallorhinchus milii (partial IPK from draft genome)Danio rerioOryzias latipesTakifugu rubripesTetraodon nigrovidrisGasterosteus aculeatusInvertebrate chordatesBranchiostoma floridaeCiona intestinalisSaccoglossus kowalevskiiCiona savignyiEchinoderms (Deuterostomes)Paracentrotus lividus (EST) (almost complete assembly)Strongylocentrotus purpuratus (almost complete prediction)ArthropodsHomarus americanus (EST)Drosophila sp. (Cassera et al., 2004)Apis melliferaAedes aegyptiAnopheles gambiaeCulex quinquefasciatusPediculus humanusIxodes scapularisDaphnia pulexOther bilateriansHirudo medicinalis (EST)Capitella teletaEisenia fetida (EST)Helobdella robustaLottia giganteaSchistosoma mansoniAplysia kurodai (EST)Schistosoma japonicumAplysia californica (partial prediction)Brugia malayiMeloidogyne incognitaPristionchus pacificusCnidariansAcropora palmate (EST)Hydra magnapilliataMontastraea faveolata (EST)Nematostella vectensis (predicted)Other early metazoansTrichoplax adherensAmphimedon queenslandicaPre-metazoans (within Holozoa)Monosiga brevicollisSalpingoeca rosettaCapsaspora owczarzakiFungiSpizellomyces punctatus34 fungal genomesRhizopus oryzaeMucor circinelloidesEpichloe festucae (EST)‡Green plantsall plants, including:Selaginella moellendorffiiPhyscomitrella patensOstreococcus tauriOstreococcus lucimarinusMicromonas pusillaChlamydomonas reinhardtiiVolvox carteriAmoebozoaDictyostelium discoideumPolysphondylium pallidumEntamoeba hislolytica§Entamoeba invadens§Entamoeba dispar§AlveolataPerkinsus marinusPlasmodium sp. (Lynen, 1967)Cryptosporidium sp. (Hedden and Kamiya, 1997)Tetrahymena thermophilaParamecium tetraureliaIchthyophthirius multifilisDiatomsThalassiosira pseudonanaPhaeodactylum tricornutumKinetoplastidaTrypanosoma sp. (Chew and Bryant, 2007)Leishmania sp. (Chew and Bryant, 2007)ExcavatesGiardia lambliaTrichomonas vaginalisOthersMalawimonas jakobiformis (EST)Naegleria gruberiAlexandrium tamarense * EST stands for expressed sequence tag. All other IPKs were found in draft or complete genomes. ‡ Since IPK was not found in related fungal genomes, this may be a case of horizontal transfer or even EST library contamination. § Greater similarity to IPK from a clade of archaea than to eukaryotes. IPK appears to have been lost independently in many animal lineages (Figure 2, Table 1). It is absent from choanoflagellates and sponges, but found in early branching animals such as Trichoplax, cnidarians (Nematostella, but not Hydra), and corals. It is found in bilaterians, including molluscs (Aplysia sp., Lottia gigantea), annelids (earthworm and leech), and a crustacean (lobster), but not in any insect or nematode. Within deuterostomes, it is found in the sea urchin and sea star, as well as the hemichordate Saccoglossus kowalevskii (acorn worm) and the chordate Branchiostoma floridae (lancelet). Within the vertebrates, it is found in a shark (Callorhinchus milii) but no teleost fish; in an amphibian (the newt Notophthalmus viridescens) but not in frogs; and in a lizard (Anolis carolinensis) and a snake (Philodryas olfersii), but not in any bird or mammal. Within the fungi, IPK is present in one of two sequenced chytrids and two other basal fungal genomes, but is otherwise absent. While all identified IPK homologs retain 11 invariant residues (see Table 2), their sequences are otherwise highly divergent, and their predicted phylogeny agrees only in part with organismal taxonomy. For instance, while all plant and animal IPKs cluster together, sequences from the archaeal class Thermoprotei fall into four distinct clusters on the calculated phylogenetic tree, and in the eukaryotic genus Entamoeba, sequences cluster with the archaeal class Methanomicrobia (Figure 2). This sporadic grouping pattern suggests horizontal gene transfer and/or parallel evolution is not uncommon and may relate to shared organismal needs in similar ecological niches. Table 2 Conserved residues in IPK https://doi.org/10.7554/eLife.00672.008 Highly-conserved residues*Partially conserved residuesK6, G8, G9, K15, G54, H60, P140, G144, D213, T215, G216K221†, G253‡, T254‡ * Numbering is in accordance with IPK from M. jannaschii. † Conserved in nearly all species except one possibly due to a gene prediction error. ‡ May be invariant, but mis-predicted in several sequences. Kinetic characterization of IPKs, PMKs, and MDDs To confirm the phylogenetic analyses and identification of putative IPK genes, we next overexpressed, purified, and biochemically characterized seven IPK homologs selected from all three domains of life. These include the previously characterized IPK from M. jannaschii (Grochowski et al., 2006; Dellas and Noel, 2010) and homologs from two other archaeal species, Methanococcus maripaludis and Sulfolobus solfataricus (one of the few archaeal species that also encodes the complete classical MVA pathway), the bacterium Roseiflexus castenholzii (an organism with an annotated MDD but no obvious PMK), and three eukaryotes, Trichoplax adhaerens (early-branching metazoan), Branchiostoma floridae (chordate), and Arabidopsis thaliana (plant). Kinetic experiments were performed using a quantitative lactate dehydrogenase-pyruvate kinase coupled assay demonstrating that all seven IPK homologs catalyze the efficient phosphorylation of IP (4) to IPP (1) with high specificity as reported in Table 3. Table 3 Steady-state kinetic constants for enzymes of the MVA pathway* https://doi.org/10.7554/eLife.00672.009 Organismgi numberEnzymeSubstrateKM (μM)kcat (s−1)Ki (μM)kcat/KM (s−1μM−1)R2 (†)M. jannaschii15668214IPKIP4.3 (± 0.6)‡1.46 (± 0.03)–§0.34 (± 0.05)0.90M. maripaludis132664414IPKIP21.4 (± 4.3)15.2 (± 1.4)877 (± 550)0.71 (± 0.16)0.99S. solfataricus15897030IPKIP23.6 (± 4.8)0.91 (± 0.05)–0.04 (± 0.01)0.9215899698PMKMVAP23.1 (± 3.8)1.98 (± 0.10)5500 (± 2200)0.09 (± 0.01)0.98R. castenholzii156743980IPKIP4.3 (± 0.7)1.70 (± 0.04)–0.40 (± 0.06)0.96156740939MPD#MVAP152 (± 38)1.7 (± 0.1)–0.011 (± 0.003)0.98156740939MDDMVAPPND#NDNDNDNDB. floridaeNA¶IPKIP13.3 (± 2.0)27.2 (± 1.2)2820 (± 1700)2.05 (± 0.32)0.98260829481PMKMVAP38.0 (± 7.3)12.6 (± 0.4)–0.33 (± 0.06)0.93260794527MDDMVAPP15.1 (± 3.7)1.36 (± 0.09)–0.09 (± 0.02)0.89A. thaliana22329798IPKIP0.79 (± 0.35)1.9 (± 0.2)522 (± 381)2.4 (± 1.1)0.9515222502PMKMVAP11.8 (± 2.0)20.9 (± 0.7)–1.77 (± 0.31)0.9715224931MDDMVAPP15.7 (± 5.0)2.02 (± 0.13)–0.13 (± 0.04)0.89T. adhaerens195996013IPKIP3.1 (± 1.6)2.4 (± 0.2)–0.77 (0.40)0.79 * See Table 3—source data 1, Table 3—source data 2, Table 3—source data 3, and Table 3—source data 4 for Michaelis-Menten fitted kinetic curves for each enzyme. † R2 represents goodness of fit for the kinetic curve to the experimental data. R2 = = of = of of the each and a horizontal through the of all ‡ in of for each calculated kinetic § was not calculated or was not floridae IPK sequence was predicted from of the genome et al., using and Table 3—source data 1 Steady-state kinetic for IPKs each Download Table 3—source data 2 Steady-state kinetic for each Download Table 3—source data 3 Steady-state kinetic for MDDs each Download Table 3—source data 4 kinetic for MPD from R. castenholzii. Download These IPK-bearing organisms all appear from to encode genes associated with the classical MVA pathway. To possible MVA pathway bifurcation, we next overexpressed, purified, and biochemically characterized and proteins from organisms biochemically from floridae and thaliana catalyze the efficient phosphorylation of MVAP as annotated (3) with similar kinetic constants PMK from solfataricus is catalytically active using ATP and MVAP (3) as however, its MDD the phosphorylation-dependent decarboxylation of MVAPP to IPP (1) at a we were to kinetic for solfataricus Kinetic characterization of a Chloroflexi MPD Unexpectedly, the MDD homolog from R. castenholzii does not catalyze the decarboxylation of MVAPP to IPP (1) at a as it the phosphorylation-dependent decarboxylation of MVAP (3) to IP (4) This annotated R. castenholzii MDD, in in a completely unexpected way as a MPD the metabolic of the MPD of the alternative MVA pathway. The catalytic constants of R. castenholzii MPD with MVAP suggest it is catalytically efficient than MDD enzymes R. castenholzii, however, is a bacterium and at its growth and et al., were not possible due to of the coupled assay et al., demonstrate that R. castenholzii MPD is at of the of the curves = at that the enzyme at well what it in and is most active at high (Figure Figure 3 Download asset Open asset of Roseiflexus castenholzii for R. castenholzii MPD in from to to in the of the as in for each of the curves show in and as a function of R. castenholzii MPD was from to at now To the of our this is the first enzyme discovered to function as a catalytically efficient the associated with functional sequence this newly discovered MPD activity encoded by an sequence suggests that functional of the enzymes the canonical MVA pathway be of alternative and classical MVA To confirm that the functional R. castenholzii MPD protein the of MVAP (3) to IP (4) in a pathway specific with a we next each pathway in using a reaction of enzymes and MVAP (3) and ATP were for with MPD and IPK from R. castenholzii. The reaction was then into a all necessary enzymes to a higher order isoprenoid the reactions were performed for the in of the classical MVA pathway using PMK and MDD from each The in classical MVA pathway reactions for and floridae and of MVAP (3) to (Figure The in classical MVA pathway reaction from R. castenholzii floridae PMK to complete the missing then to (Figure In the in alternative MVA pathway from R. castenholzii enzymes a of MVAP (3) to and solfataricus were for a possible alternative MVA pathway using their IPKs and These reactions do not any that these MDDs as from their gene and lack MPD reactions enzyme, or MVAP (3) do not any Figure 4 Download asset Open asset In and of the alternative and classical MVA pathways. steps of the MVA shown with enzymes of the alternative and classical as The putative of MDD to MPD is highlighted by a In include either the alternative or the classical enzymes to produce IPP (1) shown in as well as all including isopentenyl phosphate to produce diphosphate to produce diphosphate and to produce the product, are by and by and used are highlighted in type. Results of the in of enzyme The of the represents of enzymes shown in and and the to the end product, shown as the of organisms are as = Branchiostoma floridae At = Arabidopsis = Sulfolobus and = Roseiflexus castenholzii. is both and blue on the on it is as an MDD or an MPD of R. castenholzii MPD an MVAP The experiments that the enzyme from R. castenholzii does not MVAPP as a R. castenholzii as an MPD and a newly discovered biochemical activity, the of MVAP (3) as a for the phosphorylation-dependent decarboxylation of MVAP (3) to IP These biochemical results demonstrate that this gene in fact encodes an of sequences and of MDDs catalytic elements that MPD from R. castenholzii from other bacterial of R. castenholzii MPD was using et al., 2006; et al., and the MDD as a sequence identity to R. castenholzii et al., the diphosphate group of to MDD et al., were then with the residues in the R. castenholzii MPD of the residues of the canonical MDD from to the same in R. castenholzii MPD that the R. castenholzii MPD several conserved interactions associated with of the terminal phosphate on the diphosphate of MVAPP (Figure 5). the R. castenholzii MPD contains additional residues including and that appear to the phosphate of MVAP these to MDDs such as the R. castenholzii and with the terminal phosphate on the diphosphate of MVAPP (Figure Table Figure