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Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2 tm1Cje ). The Sod2 mutant mice exhibit a tissuespecific inhibition of the respiratory chain enzymes NADHdehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

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.

Mevalonate kinase catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the pathways of isoprenoids and sterols. Deficiency in mevalonate kinase activity has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). The crystal structure of rat mevalonate kinase in complex with MgATP has been determined at 2.4-Å resolution. Each monomer of this dimeric protein is composed of two domains with its active site located at the domain interface. The enzyme-bound ATP adopts an anti conformation, in contrast to the syn conformation reported for Methanococcus jannaschii homoserine kinase. The Mg 2؉ ion is coordinated to both ␤-and ␥-phosphates of ATP and side chains of Glu 193 and Ser 146 . Asp 204 is making a salt bridge with Lys 13 , which in turn interacts with the ␥-phosphate. A model of mevalonic acid can be placed near the ␥-phosphoryl group of ATP; thus, the C5 hydroxyl is located within 4 Å from Asp 204 , Lys 13 , and the ␥-phosphoryl of ATP. This arrangement of residues strongly suggests: 1) Asp 204 abstracts the proton from C5 hydroxyl of mevalonate; 2) the penta-coordinated ␥-phosphoryl group may be stabilized by Mg 2؉ , Lys 13 , and Glu 193 ; and 3) Lys 13 is likely to influence the pK a of the C5 hydroxyl of the substrate. V377I and I268T are the most common mutations found in patients with HIDS. Val 377 is located over 18 Å away from the active site and a conservative replacement with Ile is unlikely to yield an inactive or unstable protein. Ile-268 is located at the dimer interface, and its Thr substitution may disrupt dimer formation.Mevalonate kinase (MK, ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) 1 catalyzes the transfer of the ␥-phosphoryl group from ATP to the C5 hydroxyl oxygen of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the biosynthetic pathway for isoprenoids and sterols from acetate. Although the enzyme was discovered in the late 1950s (1, 2), it suffered over three decades of neglect, as research on the isoprenoid pathway was focused on HMG-CoA reductase, which catalyzes the previous step in the pathway, i.e. the formation of mevalonic acid from HMG-CoA. However, interest in MK has been revived recently, because it was recognized that this enzyme, together with HMG-CoA synthase and HMG-CoA reductase, is involved in coordinate regulation of this pathway and therefore may represent a secondary control point. The recent recognition of the involvement of the diverse non-sterol isoprenoid metabolites in various cellular functions (e.g. protein prenylation, protein glycosylation, and cell cycle regulation) has also increased interest in this enzyme. In addition, the significance of MK has been further highlighted by the implication of the enzyme in human inherited diseases, such as mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS, Mendelian Inheritance in Man 260920).The enzyme is found in eukaryotes, archaebacteria, and some eubacteria. Th...

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SUMMARY Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a “synthetic lethal” interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E “rewires” metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.

cDNA encoding human mevalonate kinase has been overexpressed and the recombinant enzyme isolated. This stable enzyme is a dimer of 42-kDa subunits and exhibits a V m ‫؍‬ 37 units/mg, K m(ATP) ‫؍‬ 74 M, and K m(DL-MVA) ‫؍‬ 24 M. The sensitivity of enzyme to watersoluble carbodiimide modification of carboxyl groups prompted evaluation of four invariant acidic amino acids (Glu-19, Glu-193, Asp-204, and Glu-296) by site-directed mutagenesis. Elimination of Glu-19's carboxyl group (E19A, E19Q) destabilizes the enzyme, whereas E19D is stable but exhibits only ϳ2-fold changes in V m and K m values. E296Q is a stable enzyme, which exhibits kinetic parameters comparable to those measured for wild-type enzyme. E193A is a labile protein, whereas E193Q is stable, exhibiting >50-fold diminution in V m and elevated K m values for ATP (ϳ20-fold) and mevalonate (ϳ40-fold). Such effects would be compatible with a role for Biosynthesis of isoprenoids and sterols requires the ATP-dependent phosphorylation of mevalonic acid. In humans, diminished activity of mevalonate kinase (EC 2.7.1.36), the enzyme that catalyzes this reaction (1), results in mevalonic aciduria (2). The enzyme has long been considered to be a cytosolic protein, but recent work on mevalonate kinase has implicated it in peroxisomal metabolism (3). In this context, depressed mevalonate kinase activity has also been correlated with peroxisomal deficiency disorders (4, 5). Although these observations suggest that detailed information on this enzyme would be useful, mevalonate kinase has not received as much attention as other enzymes in the isoprenoid/sterol biosynthetic pathway.Enzymological characterization of mammalian mevalonate kinase has included kinetic work (6), which suggests that the enzyme catalyzes an ordered sequential reaction with mevalonic acid assigned as the first substrate bound and phosphomevalonate as the first product released. Inhibition of the enzyme by geranyl pyrophosphate (7) and farnesyl pyrophosphate (8), metabolites that are formed downstream in the isoprenoid biosynthetic pathway, has been reported, and such inhibition has been suggested to have physiological relevance (9, 10). Although an amino acid substitution that presumably accounts for human mevalonic aciduria has been documented (11), little is known about the active site amino acids that are important to enzyme function. Group-specific reagents have been employed to demonstrate that mevalonate kinase contains reactive cysteine (6) and lysine (12) residues. Recently, the first identification of an active site amino acid was accomplished when protein chemistry and mutagenesis work indicated that lysine-13 influences ATP binding (13).Availability of a recombinant form of human mevalonate kinase would facilitate studies on inherited mutations in this enzyme. Such an enzyme, available in a stable, highly purified form and in substantial amounts, could also be useful for investigation of the structure/function correlations that account for phosphomevalonate production or for feedbac...

The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Grampositive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 Å resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 Å resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Å resolution). Comparison of these structures provides a physical basis for the significant differences in K i values observed for these inhibitors. Inspection of enzyme/ inhibitor structures identified the side chain of invariant Ser 192 as making potential contributions to catalysis. Significantly, Ser 3 Ala substitution of this side chain decreases k cat by ϳ10 3 -fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 Å cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.The ever-growing trend among many bacterial pathogens toward antibiotic resistance represents one of the single greatest threats to public health in both developing and modern nations. In particular, a growing body of literature from the last decade has demonstrated that many strains of the widespread Gram-positive organisms Staphylococcus aureus and Staphylococcus epidermidis are now insensitive toward an array of the ␤-lactam class antibiotics that were once considered frontline therapeutics (1, 2). As recently as a few years ago, the problem of antibiotic resistance was associated primarily with those infections arising from within the healthcare setting. However, recent studies have shown that resistant strains are now spreading rapidly within the community, where they may cause potentially life-threatening illness in persons not recently hospitalized or undergoing invasive medical procedures (1, 3). Given the limited nature of effective therapeutic tools to combat these diseases, all such infections must be carefully managed to prevent further spread throughout the population. As a consequence, there is now renewed interest in novel classes of antimicrobials that are effective against sensitive and...