Discovery of a metabolic alternative to the classical mevalonate pathway

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.

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

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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.

The Chloroflexi are considered to be one of the oldest phyla of photosynthetic organisms (Mulkidjanian et al., 2006, Hohmann-Marriott and Blankenship, 2011). As photoheterotrophs, Chloroflexi use light for energy but cannot fix carbon dioxide as their primary source of carbon (Bryant and Frigaard, 2006). Of the six bacterial phyla reported to contain MVA pathway-bearing organisms, only the Chloroflexi contain IPK (Lombard and Moreira, 2011), but lack an obvious PMK. Although it appears from genome annotations that the Chloroflexi encode two incomplete branches of the essential MVA metabolic pathway, we demonstrate in one Chlorflexi bacterium that this MDD-like enzyme in fact acts catalytically as a bona fide MPD. MPD and IPK from this Chloroflexi bacterium, R. castenholzii, complete the assembly of a hitherto unforeseen alternative MVA pathway that catalyzes sequential MVAP (3) decarboxylation and IP (4) phosphorylation resulting in the central hub metabolite of all organisms, the five-carbon isoprenoid building block IPP (1).

The consequences of the neofunctionalization of MDD to MPD are particularly noticeable in the substrate binding regions surrounding the diphosphate moiety of MVAPP (5) in canonical MDDs. The majority of the residues surrounding the diphosphate of MVAPP (5) in canonical MDDs are strictly conserved in structure- and sequence-based analyses (Byres et al., 2007). On the other hand, archaeal MDDs from Haloarchaea and Thermoplasmata contain MPD-like active site residues associated with MVAP (3) binding and turnover and their protein sequences cluster with the MPD from Chloroflexi (Table 4) (Lombard and Moreira, 2011). These MDDs belong to two archaeal classes that encode IPK but are missing PMK, suggesting that they also use this metabolic assembly of the alternative MVA pathway. The correlation between unique active site residues and alteration in substrate preference, MVAP (3) over MVAPP (5), among MPDs from Chloroflexi bacteria, reflects a lineage specific adaptation that allows for the utilization of the alternative MVA pathway; this adaptation may also apply to MPD-like MDDs of the archaeal phyla Haloarchaea and Thermoplasmata but awaits experimental verification currently in progress.

Despite key amino acid differences in the active site of the MDD-annotated R. castenholzii MPD, it nevertheless possesses high sequence similarity to canonical MDDs. Moreover, R castenholzii MPD and its closely related homologs annotated in several sequenced Chloroflexi species’ genomes including, Roseiflexus sp. RS-1, Herpetosiphon aurantiacus, Chloroflexus aggregans, Chloroflexus aurantiacus, and Chloroflexus sp. Y-400-fl (http://www.genome.jp/kegg-bin/show_pathway?map00900), are all labeled as MDDs but share the alternative active site features of R. castenholzii MPD. Unexpectedly, this R. castenholzii gene, which is annotated as an ‘MDD’, catalyzes the phosphorylation-dependent decarboxylation of MVAP (3), thus serving as a long sought MPD requiring functional re-annotation in R. castenholzii and most likely the additional Chloroflexi species noted previously. It is possible that this enzyme has only recently emerged and undergone neofunctionaliation to acquire MPD activity from an MDD ancestor within the larger phylum of Chloroflexi heterotrophic photosynthetic bacteria. The emergence of this MPD activity may have also coincided with the loss of the PMK gene from species within the narrower Chloroflexi class and/or acquisition of an IPK gene. Selection for such an alternative metabolic route to the core isoprenoid building block IPP may be due, in part, to a higher demand for metabolic flux through an alternative set of enzymes associated with the Chloroflexi MVA pathway. Indeed, the Chloroflexi class of bacteria appear to possess an expanded repertoire of downstream enzymes of isoprenoid metabolism suggesting a high demand for isoprenoid metabolites (http://www.genome.jp/kegg-bin/show_pathway?map00900).

While archaeal species contain an active IPK consistent with the presence of the alternative MVA pathway, most lack typical or atypical MDD genes; furthermore, a gene candidate capable of encoding an enzyme able to convert MVAP (3) to IP (4), the first postulated step of the alternative pathway, has not been identified. One candidate gene previously suggested to encode this decarboxylation activity is a gene encoding a dioxygenase-like protein that resides within the MVA operon in most Archaea (MJ0403 in M. jannaschii) (Grochowski et al., 2006). MJ0403 encodes a protein that is similar in sequence to subunit B of class III extradiol ring-cleavage dioxygenases and is also homologous to the human protein MEMO (mediator of erbB2-driven cell motility). Thus far, in vitro attempts to demonstrate decarboxylase activity for MJ0403 have failed to show any turnover of MVAP (3) to IP (4) even though the protein is quite easily produced heterologously.

While most archaea lack the MDD gene, certain species from the archaeal order Sulfolobales (including the archaeon S. solfataricus) encode a gene annotated as MDD. In our experiments, S. solfataricus PMK and MDD activities were detected in steady-state kinetic experiments and GC-MS assays, respectively (Table 3, Figure 4). A recent publication on the characterization of the classical MVA pathway enzymes from crude extracts of S. solfataricus supports the presence of an active classical MVA pathway in the order Sulfolobales, consistent with our in vitro results (Nishimura et al., 2013). While our experiments demonstrate that IPK is active in an in vitro assay, their experiments involving cell-free extracts of S. solfataricus indicate that IPK activity is undetectable. Nevertheless, these combined results apply to only a small set of Archaea, and the question remains as to how most archaeal species convert MVAP (3) to the essential metabolite, IPP (1).

Eukaryotic genomes examined to date appear to encode a classical MVA pathway with few exceptions (Cassera et al., 2004). We did however identify a spotty distribution of eukaryotes that contain IPK, and from those examined thus far, corresponding IPK activity that could be associated with an alternative route to IPP (1) through the MVA pathway. Since many eukaryotes encode putative enzymes of unknown function, they may also encode a cryptic MPD that would serve in an alternative biosynthetic route to IPP (1). All IPKs tested thus far were fully functional, demonstrating that true IPKs persist across most eukaryotic lineages, while some have been lost during rare evolutionary events, probably due to partial redundancy with the MVA pathway. The unusual phylogeny of IPK coupled with its membership in a family of kinases that phosphorylate such a broad range of substrates leave open the possibility that IPK may assume varied physiological roles, including phosphorylation of an IP-like substrate, IP recycling, and/or most notably, access to a pool of IPP through IP-IPP homeostatic control.

While the IPK gene is present within a spotty distribution of eukaryotes, the gene appears to be universally retained across the green plant lineage, which suggests that it plays a more universal role within the plant kingdom. Plants are unique in that they are currently known to encode two IPP-synthesizing pathways, the DXP pathway localized to the chloroplast and the MVA pathway localized to the peroxisome and cytoplasm (Lange et al., 2000, Leivar et al., 2005; Nagegowda et al., 2005; Hsieh et al., 2008). These pathways assume distinct metabolic roles within general isoprenoid biosynthesis (Nes and Venkatramesh, 1999; Eisenreich et al., 2001). It would not be surprising to identify yet another variation of the MVA pathway in the green plant lineage. In addition to IPP (1) recycling through IP (4), IPK would afford metabolic control of carbon availability from IP (4) to IPP (1). Noting that the diversity of primary and secondary isoprenoid products produced by plants often localize to subcellular compartments, organelles and specialized cell types (i.e. trichomes), an ubiquitous IPK in plants may regulate spatial and temporal control of isoprenoid diphosphate metabolism destined for a myriad of primary and specialized plant metabolites.

These results biochemically elucidate the terminal biosynthetic steps in an alternative or unconventional MVA pathway. The experiments described also definitively demonstrate metabolic routes to IPP (1) through the classical MVA pathway in previously overlooked organisms by including the kinetic characterization of IPKs, MDDs, MPDs, and PMKs taken from all three domains of life. Significantly, the studies presented provide the first experimental support for the existence of IPK catalytic activity in all three domains of life and a fully functional alternative MVA pathway in the photosynthetic heterotrophic bacterial class Chloroflexi.

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Author details

1. Nikki Dellas

   1. Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, United States
   2. Jack H Skirball Center for Chemical Biology and Proteomics, Salk Institute for Biological Studies, La Jolla, United States
   3. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, United States

   Contribution

   ND, Conceived and completed the project including all experimental designs and implementations, deep phylogenetic analyses under the supervision of Gerard Manning, analyzed all data, drafted the complete article, and revised the article in response to reviewers’ comments

   Competing interests

   The authors declare that no competing interests exist.

2. Suzanne T Thomas

   Jack H Skirball Center for Chemical Biology and Proteomics, Salk Institute for Biological Studies, La Jolla, United States

   Contribution

   STT, Carried out the measurements of the pH-dependent thermostability of MPD, carried out steady-state kinetic measurements on purified MPD with its initial affinity tag removed, and edited near final versions of the article

   Competing interests

   The authors declare that no competing interests exist.

3. Gerard Manning

   Razavi Newman Center for Bioinformatics, Salk Institute for Biological Studies, La Jolla, United States

   Present address

   Department of Bioinformatics and Computational Biology, Genentech, San Francisco, United States

   Contribution

   GM, Instructed Nikki Dellas on methods and interpretations of deep phylogenetic analyses described in the article, carried out computational methods described, provided an evolutionary context for the article, and together with Nikki Dellas drafted the initial article and edited the initially submitted article

   For correspondence

   manning@manninglab.org

   Competing interests

   The authors declare that no competing interests exist.

4. Joseph P Noel

   1. Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, United States
   2. Jack H Skirball Center for Chemical Biology and Proteomics, Salk Institute for Biological Studies, La Jolla, United States

   Contribution

   JPN, Directed the project including experimental planning with Nikki Dellas, assisted in data analysis, interpreted data, drafted sections of the article together with Nikki Dellas and provided editing at all stages

   For correspondence

   noel@salk.edu

   Competing interests

   The authors declare that no competing interests exist.

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