Mevalonate Pathway Of Isoprenoid Biosynthesis Research Articles

A computational study of the molecular basis of antibiotic resistance in a DXR mutant.

Proteins of the independent mevalonate pathway for isoprenoid biosynthesis are important targets for the development of new antibacterial compounds as this pathway is present in most pathogenic organisms such as Mycobacterium tuberculosis, DPlasmodium falciparum and Escherichia coli, but is not present in mammalian species, including humans. Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an important target in this pathway and the most effective DXR inhibitor to date is fosmidomycin, which is used to treat malaria and, more recently, tuberculosis. Recently, Armstrong C. M. et al. showed that a mutant of DXR, S222T, induces a loss of the fosmidomycin inhibition efficiency, even though the bacteria culture is still viable and able to produce isoprenoids. As this represents a potential fosmidomycin-resistant mutation, it is important to understand the mechanism of this apparent mutation-induced resistance to fosmidomycin. Here, we used molecular dynamics simulations and Molecular Mechanics/Poisson Boltzmann Surface Area analysis to understand the structural and energetic basis of the resistance. Our results suggest that the point mutation results in changes to the structural dynamics of an active site loop that probably protects the active site and facilitates enzymatic reaction. From the simulation analysis, we also showed that the mutation results in changes in the interaction energy profiles in a way that can explain the observed activity of the mutant protein toward the natural inhibitor deoxy-D-xylulose 5-phosphate. These results should be taken into consideration in future efforts to develop new therapeutic antibiotic compounds that target DXR.

Sequence Analysis of HMG-CoA Synthase Like Gene from Brassica rapa

Terpenoids are synthesized in plants through the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways, among isopentenyl diphosphate (IPP) as the central intermediate metabolite. 3-Hydroxy-3-methylglutaryl- Coenzyme A synthase (HMGS) is a subsequent enzyme in MVA pathway of isoprenoid biosynthesis and catalyses the condensation of acetyl-CoA by acetoacetyl-CoA to yield HMG-CoA. Different genes of HMGS are present in different species of plants and animals for the synthesis of secondary metabolites. The present study was planned to compare and analyse bioinformatically the HMG-CoA gene sequence of Brassica rapa with other sequences of different species. The gene sequence have 925 bp coding DNA sequence and was submitted in National Centre for Biotechnology Information (NCBI) with accession Number. MF577077. The alignment of nucleotide and protein sequences was carried out through Geneious. Bioinformatics comparison including structural and functional analysis of HMG-CoA synthase like gene sequence isolated from Brassica rapa was carried out by using different bioinformatics tools and softwares. These comparisons gave a better information about properties, genetic and functional diversity of this gene along with amino acid substitutions.

Efficient Production of δ-Guaiene, an Aroma Sesquiterpene Compound Accumulated in Agarwood, by Mevalonate Pathway-Engineered <i>Escherichia coli</i> Cells

Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ-guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 - 42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engineered E. coli cells showed an appreciable δ-guaiene producing activity in the en- riched medium in the presence of appropriate isoprene precursors.

Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells.

Past achievements, current status and future perspectives of studies on 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) in the mevalonate (MVA) pathway

HMGS functions in phytosterol biosynthesis, development and stress responses. F-244 could specifically-inhibit HMGS in tobacco BY-2 cells and Brassica seedlings. An update on HMGS from higher plants is presented. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is the second enzyme in the mevalonate pathway of isoprenoid biosynthesis and catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce S-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Besides HMG-CoA reductase (HMGR), HMGS is another key enzyme in the regulation of cholesterol and ketone bodies in mammals. In plants, it plays an important role in phytosterol biosynthesis. Here, we summarize the past investigations on eukaryotic HMGS with particular focus on plant HMGS, its enzymatic properties, gene expression, protein structure, and its current status of research in China. An update of the findings on HMGS from animals (human, rat, avian) to plants (Brassica juncea, Hevea brasiliensis, Arabidopsis thaliana) will be discussed. Current studies on HMGS have been vastly promoted by developments in biochemistry and molecular biology. Nonetheless, several limitations have been encountered, thus some novel advances in HMGS-related research that have recently emerged will be touched on.

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe

Isoprenoids may be synthesized via one of two pathways, the classical mevalonate pathway or the alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. While the majority of bacteria utilize a single pathway for isoprenoid biosynthesis, Listeria monocytogenes is unusual in possessing the complete set of genes for both pathways. Here, we utilized new molecular tools to create precise gene deletions in selected genes encoding enzymes of both pathways, gcpE, lytB (encoding proteins in the MEP pathway) and hmgR (encoding a protein in the mevalonate pathway). We demonstrate that the hmgR gene can only be deleted when the growth medium is supplemented with exogenous mevalonate. Furthermore, full growth of the mutant in the absence of mevalonate was only possible when the intact hmgR gene was supplied in trans using an IPTG-inducible expression system. Murine competitive index assays performed via the oral and intraperitoneal routes of infection revealed that the mevalonate hmgR mutant could not be recovered from livers and spleens 3 days post-infection. We propose that HmgR in L. monocytogenes EGDe is involved in essential metabolic functions and that an intact MEP pathway is not capable of sustaining growth.

Multilevel Control of Arabidopsis 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase by Protein Phosphatase 2A

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B'' regulatory subunits of protein phosphatase 2A (PP2A), designated B''α and B''β, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B''α and B''β are Ca²⁺ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B''α and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B''β. Our data indicate that PP2A exerts multilevel control on HMGR through the five-member B'' protein family during normal development and in response to a variety of stress conditions.

Origins and Early Evolution of the Mevalonate Pathway of Isoprenoid Biosynthesis in the Three Domains of Life

Isoprenoids are a very diverse family of organic compounds widespread in the three domains of life. Although they are produced from the condensation of the same precursors in all organisms (isopentenyl pyrophosphate and dimethylallyl diphosphate), the evolutionary origin of their biosynthesis remains controversial. Two independent nonhomologous metabolic pathways are known: the mevalonate (MVA) pathway in eukaryotes and archaea and the methylerythritol phosphate (MEP) pathway in bacteria and several photosynthetic eukaryotes. The MVA pathway is also found in a few bacteria, what has been explained in previous works by recent acquisition by horizontal gene transfer (HGT) from eukaryotic or archaeal donors. To reconsider the question of the evolutionary origin of the MVA pathway, we have studied the origin and the evolution of the enzymes of this pathway using phylogenomic analyses upon a taxon-rich sequence database. On the one hand, our results confirm the conservation in archaea of an MVA pathway partially different from eukaryotes. This implies that each domain of life possesses a characteristic major isoprenoid biosynthesis pathway: the classical MVA pathway in eukaryotes, a modified MVA pathway in archaea, and the MEP pathway in bacteria. On the other hand, despite the identification of several HGT events, our analyses support that the MVA pathway was ancestral not only in archaea and eukaryotes but also in bacteria, in contradiction with previous claims that the presence of this pathway in bacteria was due to HGT from other domains. Therefore, the MVA pathway is likely an ancestral metabolic route in all the three domains of life, and hence, it was probably present in the last common ancestor of all organisms (the cenancestor). These findings open the possibility that the cenancestor had membranes containing isoprenoids.

The PPR protein encoded by the LOVASTATIN INSENSITIVE 1 gene is involved in RNA editing at three sites in mitochondria of Arabidopsis thaliana

Post-transcriptional RNA editing in flowering plant mitochondria alters several hundred nucleotides from cytidine to uridine, mostly in mRNAs. To characterize the factors involved in RNA editing in plant mitochondria, we initiated a screen for nuclear mutants defective in RNA editing at specific sites. Here we identify the nuclear-encoded gene MEF11, which is involved in RNA editing of the three sites cox3-422, nad4-124 and ccb203-344 in Arabidopsis thaliana. A T-DNA insertion line of this gene was previously characterized as showing enhanced tolerance to the compound lovastatin, an inhibitor of the mevalonate pathway of isoprenoid biosynthesis. The mef11-1 mutant described here shows similar tolerance to lovastatin. Identification of the function of the MEF11 protein in site-specific mitochondrial RNA editing suggests indirect effects of retrograde signalling from mitochondria to the cytoplasm to evoke alteration of the mevalonate pathway. The editing sites cox3-422 and ccb203-344 each alter amino acids that are conserved in the respective proteins, while the nad4-124 site is silent. The single amino acid change in the mef11-1 mutant occurs in the second pentatricopeptide repeat, suggesting that this motif is required for site-specific RNA editing.

Molecular cloning, characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Salvia miltiorrhiza

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115 bp containing a 1,695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones biosynthesis at the molecular level.

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

Crystal structure of human phosphomavelonate kinase at 1.8 Å resolution

Interrelation between the pathways of isoprenoid biosynthesis and carbon source catabolism in anaerobic and facultatively anaerobic bacteria

Data on the interrelation between the pathways of the carbon source catabolism and isoprenoid biosynthesis in anaerobic and facultatively anaerobic bacteria were obtained. Two pathways of isoprenoid biosynthesis (nonmevalonate and mevalonate) were revealed in the representatives of the genus Clostridium. The non-mevalonate pathway of isoprenoid biosynthesis and the glycolytic pathway of substrate oxidation are typical of glucose-grown bacteria, whereas the pentose phosphate cycle operates in xylose-grown bacteria. The mevalonate pathway of isoprenoid biosynthesis was revealed in strain Clostridium thermosaccharolyticum DSM 571 grown in the presence of mevinolin, as well as in a number of lactic acid bacteria. Mevinolin is known to react with the lactate dehydrogenase complex, preventing reduction of pyruvate. The nonmevalonate pathway of isoprenoid biosynthesis was revealed in Bifidobacterium bifidum. The role of different metabolic pathways in isoprenoid biosynthesis is discussed.

HMG-CoA reductase is regulated by salinity at the level of transcription in Haloferax volcanii

The moderately halophilic archaeon Haloferax volcanii was surveyed for protein profile changes correlated with growth at high and low salinity. A single polypeptide with an approximate mass of 46 kDa was conspicuously more abundant during growth at high salinity. This protein was identified as HMG-CoA reductase (HMGR), encoded by the hmgR gene. HMGR is a key enzyme in the mevalonate pathway of isoprenoid biosynthesis, the sole route in haloarchaea for lipid and carotenoid production. Enzymatic assays confirmed that HMGR activity is more abundant in cells grown at high salinity. Low salt cultures of H. volcanii contained lower amounts of hmgR transcript compared to cells grown in high salt suggesting that the observed regulation occurs at the level of transcription. Paradoxically, both lipid and carotenoid content decreased in H. volcanii grown at high salinity despite the increased levels of HMGR specific activity. To our knowledge, this is the first report demonstrating that the expression of HMGR is regulated in response to non-optimal salinity in a halophilic archaeon.

Lipids of Archaebacteria

Summary The archaebacteria currently consist of several distinct subgroups including methanogens, extreme halophiles and certain thermoacidophiles. The lipids of archaebacteria are distinguished from those of other prokaryotes and eukaryotes by the absence of fatty acid glycerol ester lipids and the predominance of nonsaponifiable lipids. The lipid composition of the archaebacteria consists of isoprenoid and hydroisoprenoid hydrocarbons and isopranyl glycerol ether lipids. The glycerol ethers of archaebacteria, which constitute the hydrophobic residues of the polar lipids and consequently the membrane interior are diphytanylglycerol diethers or dibiphytanyldiglycerol tetraethers. Either or both glycerol ether structures may be present, depending on genus. The tetraethers of the thermoacidophilic archaebacteria are more specialized in that the dibiphytanyl alkyl chains may contain 1 to 4 cyclopentyl rings. As a consequence of the presence of the tetraethers which can span the membrane, some archaebycterial membranes may exist as a lipid “monolayer” rather than the usual lipid bilayer. The structure of some diether-containing polar lipids of archaebacteria have been well established. The extent of the variety of tetraether containing polar lipid structures is still largely unknown, but both the symmetric and asymmetric substitution of polar head groups to the tetraether has been established in some instances. Among neutral lipids, squalenes and isoprenoid hydrocarbons appear to be universal. The exact pathways for the biosynthesis of the lipid components remain a challenge, but clearly the mevalonate pathway for isoprenoid biosynthesis is the major route of lipid synthesis in archaebacteria rather than the malonyl-CoA pathway for fatty acid biosynthesis in prokaryotes and eukaryotes. The isopranyl glycerol ethers are distinctive, providing a useful taxonomic tool and molecular marker for the identification of archaebacteria. The lipids can also serve as useful biochemical “fossil” evidence for tracing the earlier existence of the organisms. Overall, the discontinuity of archaebacterial lipids formulates a point for delineating early stages of biological evolution and supports the concept that archaebacteria represent a third line of evolutionary descent.