Isoprenoids are a large and diverse class of compounds containing more than 40,000 naturally occurring terpenes and terpenoids (33
). They encompass many classes of bioactive molecules, including carotenoids, steroid hormones, phytols, redox carriers, secondary metabolites, and pheromones, that make them commercially attractive for the production of compounds varying from pharmaceuticals to biofuels (5
). Currently, a number of groups are working on increasing the production of terpenoid compounds for a variety of medicinal, agricultural, sustainable biofuel, and biomaterial applications (21
). All isoprenoids are biosynthesized from the five carbon precursors, isopentenyl diphosphate (IPP), and its isomer, dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of these central metabolites have been described, the mevalonate pathway (28
) and the 2-C-methyl-d
-erythritol 4-phosphate (MEP) pathway (25
). The mevalonate pathway typically is found in animals, plants, and in many Gram-positive bacteria, including Streptococcus pneumoniae
). Some enzymes of the mevalonate pathway also have been identified in archaea; however, the complete pathway has not been elucidated (27
). The mevalonate pathway catalyzes the conversion of three molecules of acetyl coenzyme A (CoA) to IPP and DMAPP. Briefly, two molecules of acetyl-CoA undergo a Claisen condensation to form acetoacetyl-CoA, which is catalyzed by acetoacetyl-CoA thiolase. 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase then catalyzes an aldol reaction between acetoacetyl-CoA and a third molecule of acetyl-CoA. The conversion of HMG-CoA to mevalonate subsequently is catalyzed by HMG-CoA reductase. Mevalonate kinase (MVK) and phosphomevalonate kinase (PMK) catalyze the phosphorylation of the primary alcohol of mevalonate and the phosphate of phosphomevalonate, respectively, to form diphosphomevalonate. The penultimate reaction in the pathway is the phosphorylative decarboxylation of diphosphomevalonate catalyzed by the diphosphomevalonate decarboxylase to yield IPP (10
). IPP isomerase (IDI) catalyzes the conversion of IPP to DMAPP ().
Fig. 1. Mevalonate pathway. The proposed modified pathway for the production of isoprenoids in archaea organisms is illustrated in the box (17).
A distinguishing characteristic of archaeal organisms is that isoprenoids make up the major component of their membrane lipids. In contrast, the lipids of eukaryotic and bacterial organisms are composed primarily of fatty acids (6
). Studies of isoprenoid biosynthesis in archaea have demonstrated that both acetate and mevalonate are precursors for IPP formation, indicating that the mevalonate pathway is involved in their biosynthesis (11
). Putative homologues of all mevalonate pathway genes, excluding the diphosphomevalonate decarboxylase, have been identified in archaea by genomic analysis (3
). In addition, putative isopentenyl monophosphate kinases have been identified and characterized from archaea, suggesting the possible utilization of a modified mevalonate pathway for the production of isoprenoids in archaea (8
Eukaryotic, bacterial, and archaeal organisms must ensure the sufficient production of a variety of isoprenoid compounds that are essential for the proper growth, signaling, transport, and life cycle controls as well as the prevention of the overaccumulation of potentially toxic products, such as cholesterol (15
). Organisms manage these tasks through the intricate regulation of isoprenoid-producing pathways (15
). MVK was demonstrated to be an important regulatory point in the mevalonate pathway in both bacteria (1
) and eukaryotes (4
). Previously the small-molecule regulation of MVKs could be divided into two classes. The first class is inhibited by metabolites downstream of the diphosphomevalonate decarboxylase reaction (IPP, DMAPP, GPP, FPP, and longer chain isoprenoids) (9
). The regulation of a eukaryotic MVK isolated from pig liver was first reported by Dorsey and Porter in 1968 (9
). Their detailed kinetic analysis revealed the significant feedback regulation of this enzyme by GPP and FPP and, to a lesser degree, by DMAPP, IPP, and PPi
). Human MVK subsequently was characterized and found to be inhibited by FPP, GPP, IPP, DMAPP, and geranylgeranyl pyrophosphate (18
). The characterization of four plant MVKs and S. cerevisiae
MVK by Gray and Kekwick in 1972 revealed that they all are inhibited by GPP, FPP, geranylgeranyl pyrophosphate, and phytyl pyrophosphate (16
). In addition, two MVKs from Gram-positive cocci, Staphylococcus aureus
and Enterococcus faecalis
, were found to be competitively inhibited by FPP with respect to ATP, with a Ki
of 45 μM (31
The second class of MVKs is inhibited by diphosphomevalonate but not by metabolites downstream of the diphosphomevalonate decarboxylase. Interestingly, DMAPP, IPP, GPP, and FPP were not feedback inhibitors of the Gram-positive bacterium S. pneumoniae
MVK at concentrations of up to 12 μM; however, diphosphomevalonate inhibited S. pneumoniae
MVK at nanomolar concentrations (2
We report the overexpression, purification, kinetic analysis, and inhibition studies of the mvk gene product from the archaeon Methanosarcina mazei. The S. cerevisiae and S. pneumoniae MVKs have been recharacterized in this study and serve as positive controls for the two known classes of feedback-regulated MVKs. Our findings demonstrate that, unlike MVKs from S. cerevisiae and S. pneumoniae, M. mazei MVK is not inhibited by known feedback inhibitors of MVKs. A phylogenetic tree of 29 MVK representatives from Archaea, Eukarya, and Bacteria indicates a clear evolutionary separation of the mvk gene between these domains and leads to the hypothesis that these distinct branches utilize alternative regulation mechanisms ().
Phylogenetic tree for MVKs from the mevalonate pathway of Eukarya, Archaea, and Bacteria.
Accordingly, we conclude that there are at least three classes of MVKs that can be differentiated based on their inhibition profiles.