Diphosphomevalonate Decarboxylase Research Articles

Recent Advances and New Insights in Genome Analysis and Transcriptomic Approaches to Reveal Enzymes Associated with the Biosynthesis of Dendrobine-Type Sesquiterpenoid Alkaloids (DTSAs) from the Last Decade.

Dendrobium species, which are perennial herbs widely distributed in tropical and subtropical regions, are notable for their therapeutic properties attributed to various bioactive compounds, including dendrobine-type sesquiterpenoid alkaloids (DTSAs). The objective of this review article is to provide a comprehensive overview of recent advances in the biosynthesis of DTSAs, including their extraction from Dendrobium species and endophytes, elucidation of associated genes through genomic and transcriptomic sequencing in both Dendrobium spp. and endophytes, exploration of the biosynthetic pathways of DTSAs, and drawing conclusions and outlining future perspectives in this field. Alkaloids, predominantly nitrogen-containing compounds found in medicinal orchids, include over 140 types discovered across more than 50 species. DTSAs, identified in 37 picrotoxane alkaloids, have a distinctive five-membered nitrogen heterocyclic ring. This review highlights endophytic fungi as alternative sources of DTSAs, emphasizing their potential in pharmaceutical applications when plant-derived compounds are scarce or complex. Genomic and transcriptomic sequencing of Dendrobium spp. and their endophytes has identified key genes involved in DTSAs biosynthesis, elucidating pathways such as the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways. Genes encoding enzymes, such as acetyl-CoA C-acetyltransferase and diphosphomevalonate decarboxylase, are positively associated with dendrobine production. Despite significant advancements, the complexity of terpenoid biosynthesis in different subcellular compartments remains a challenge. Future research should focus on leveraging high-quality genomic data and omics technologies to further understand and manipulate the biosynthetic pathways of DTSAs and enhance their medicinal use.

266 Interactive Effects of Dietary Isoleucine and Valine Ratios to Lysine in Response to Varying Leucine to Lysine Ratios on Hepatic Protein Expression in Commercial Broilers

Abstract A central composite design (CCD) study was conducted to understand the relationship among dietary branched chain amino acid (BCAA) ratios and expression of proteins related to amino acid and protein metabolism in the liver of broilers. A total of 2,592 d-old Ross 344 × 708 male broilers were randomly placed in 144 floor pens. Each pen received 1 of 15 dietary treatments in a 23 CCD with 6 center points from 20 to 35 d of age, varying in digestible ratios of isoleucine:lysine (Ile:Lys; 52 to 75), valine:lysine (Val:Lys; 64 to 87), and leucine:lysine (Leu:Lys; 110 to 185). On d 35, 1 bird per pen was selected, euthanized, and liver tissue samples were collected for protein extraction and proteomic analysis via data independent acquisition protein sequencing with a timsTOF Pro 2 LC/MS/MS and Spectronaut 15 software. Dihydrolipoamide acetyltransferase (PDHX), aspartate aminotransferase (GOT2), branched-chain-amino-acid aminotransferase (BCAT1), diphosphomevalonate decarboxylase (MVD), acylamino-acid-releasing enzyme (APEH), and aspartate aminotransferase (GOT1) were identified and quantified. Protein quantification data were analyzed as a CCD using the RSREG procedure of SAS v. 9.4 with significance declared at P ≤ 0.10. When the individual AA effect in the response surface (RS) regression analysis was P ≤ 0.2, raw protein quantification data were analyzed as a 1-way ANOVA and the PDIFF option of SAS set at P ≤ 0.1 using the GLIMMIX procedure. The RS model for MVD and APEH expression were significant (P ≤ 0.0511). However, models for PDHX, GOT2, BCAT1, and GOT1 were not significant (P ≥ 0.1356) and the R2 values ≤ 0.25 for all proteins did not allow for prediction of protein expression means. No linear, quadratic, cross product, or individual BCAA ratio effects were observed for PDHX, GOT2, and BCAT1 (P ≥ 0.1977). A linear model for MVD and APEH protein expression (P ≤ 0.8030) and quadratic model for APEH (P ≤ 0.0552) were significant. MVD increased as Val:Lys ratio increased when Leu:Lys and Ile:Lys were fixed at 148 and 63 or 170 and 70, respectively (P ≤ 0.0926). Low Val:Lys ratio expressed the least hepatic APEH expression when Leu:Lys and Ile:Lys ratios were 148 and 63 or 125 and 70, respectably (P ≤ 0.0153). Hepatic MVD expression decreased as Leu:Lys ratio increased when Val:Lys and Ile:Lys were 75 and 63, respectably (P ≤ 0.0044). GOT1 expression was higher with 170 Leu:Lys ratio compared with 125, when Val:Lys and Ile:Lys ratio were 68 and 70, respectably (P ≤ 0.0460). APEH was greater with a low Ile:Lys ratio when Val:Lys and Leu:Lys were 68 and 125, respectably (P ≤ 0.0786). Varying concentrations of dietary BCAA and their interactions seem to alter hepatic protein expression and may help explain the mechanisms underlying their influence on broiler growth.

Time-resolved transcriptomic profile of oleaginous yeast Rhodotorula mucilaginosa during lipid and carotenoids accumulation on glycerol

The study reports the exploration of the transcriptome landscape of the red oleaginous yeast Rhodotorula mucilaginosa IIPL32 coinciding with the fermentation kinetics of the yeast cultivated in a two-stage fermentation process to exploit the time-series approach to get the complete transcripts picture and reveal the persuasive genes for fatty acid and terpenoid synthesis. The finding displayed the molecular drivers with more than 2-fold upregulation in the nitrogen-limited stage than in the nitrogen-excess stage. The rate-limiting diphosphomevalonate decarboxylase, acetylCoA-citrate lyase, and acetyl-CoA C-acetyltransferase were significant in controlling the metabolic flux in the synthesis of reduced compounds, and acetoacetyl-CoA synthase, 3-ketoacyl-acyl carrier-protein reductase, and β-subunit enoyl reductase catalyze the key starting steps of lipids or terpenoid synthesis. The last two catalyze essential reduction steps in fatty acid synthesis. These enzymes would be the prime targets for the metabolic engineering of the oleaginous yeast for enhanced fatty acids and terpenoid production.

Comparative proteome profiling of fusarium chlamydosporum and elucidation of pigment biosynthetic pathway under nitrogen stress

The main aim of this study was to understand the protein expression of F. chlamydosporum in two different medium composition in varying concentrations of nitrogen. The interesting phenomenon of producing diverse pigments by a single strain in different concentrations of Nitrogen made us further to explore the difference in the protein expression of the fungus when grown in these two media. For this, we had adopted non-gel-based method of protein separation by LC-MS/MS analysis followed by label free identification of proteins by SWATH analysis. The molecular and biological functions of each protein and their Gene Ontology annotations were analyzed by UniProt KB and KEGG pathway; the secondary metabolite pathways and the carbohydrate metabolic pathways were analyzed by DAVID bioinformatics tool. The positively regulated proteins biologically functioned for the secondary metabolite production in optimized medium were Diphosphomevalonate decarboxylase (terpenoid backbone biosynthesis), Phytoene synthase (carotenoid biosynthesis), 6,7-dimethyl-8-ribityllumazine synthase (riboflavin biosynthesis). The main characteristic change observed was that proteins related to carotenoid biosynthesis and terpenoid synthesis were not regulated in nitrogen limited medium. Except for the protein 6,7-dimethyl-8-ribityllumazine synthase, all the enzymes related to fatty acid biosynthesis and polyketide chain elongation were up regulated. Apart from the proteins related to secondary metabolite production, two novel proteins were found to be up regulated in Nitrogen Limited Medium; C-fem protein responsible for fungal pathogenesis and DAO domain containing protein which functions as a neuromodulator and catalyzes the synthesis of dopamine. SignificanceThis particular strain of F. chlamydosporum of immense genetic and biochemical diversity represents an interesting example of a microorganism which can produce a variety of bioactive compounds and this can be exploited in various industries. The production of carotenoids and polyketides by this fungus when grown in the same media with different concentrations of Nitrogen has been published by us following which we analyzed the proteome sequence of the fungus in varying Nutrient conditions. Following the proteome analysis and expression, we could derive the pathway leading to the biosynthesis of varying secondary metabolites by the fungus which has not been published or studied so far.

Comparative Transcriptome Analysis Identifies Potentially Relevant Genes in Rubber Clones with a High Latex Yield (Hevea brasiliensis)

Since the 1950s, the rubber tree (Hevea brasiliensis) has significantly contributed to Malaysia's agricultural economy due to its capability to produce high-value natural rubber. Due to the presence of cis-1,4-polyisoprene, natural rubber exhibits unique properties such as flexibility, high elasticity, and efficient heat dispersion. Cis-1,4-polyisoprene is synthesized via two distinct metabolic pathways: the isoprenoid and rubber biosynthesis. RNA sequencing was performed on 12 specimens from the bark, leaf, and latex tissues of two rubber clones, RRIM 3001 (high latex yield) and RRIM 712 (low latex yield). After quality assessment, these specimens generated a mean of 73,816,244 clean reads. The differential expression analysis revealed that the Acetyl-CoA-Acetyltransferase (AACT) and Diphosphomevalonate Decarboxylase (MVD) genes (via the MVA pathway), as well as the Small Rubber Particle Protein (SRPP) and Rubber Elongation Factor (REF) genes (via the rubber biosynthesis pathway), were overexpressed in the bark, leaf, and latex tissues of RRIM 3001 compared to RRIM 712. The transcription factor Ethylene Response Factor (ERF) was also found to be more highly expressed in RRIM 3001 than in RRIM 712. Five rubber clones with random yield characteristics were used in quantitative amplification analysis to validate AACT, MVD, SRPP, REF, and ERF genes. The quantitative analysis shows that the five genes were consistently expressed with the yield characteristics of the five random rubber clones.

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate–binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Comparative Transcriptome and Phytochemical Analysis Provides Insight into Triterpene Saponin Biosynthesis in Seeds and Flowers of the Tea Plant (Camellia sinensis).

Triterpene saponins exhibit various biological and pharmacological activities. However, the knowledge on saponin biosynthesis in tea plants (Camellia sinensis L.) is still limited. In this work, tea flower and seed samples at different developmental stages and leaves were collected and analyzed with UPLC-PDA-MS and RNA sequencing for saponin determination and transcriptome comparison. The saponin content reached around 19% in the freshly mature seeds and 7% in the green flower buds, and decreased with the fruit ripeness and flower blooming. Almost no saponins were detected in leaf samples. PCA and KEGG analysis suggested that the gene expression pattern and secondary metabolism in TF1 and TS2 vs. leaf samples were significantly different. Weighted gene coexpression network analysis (WGCNA) uncovered two modules related to saponin content. The mevalonate (MVA) instead of 2-C-methyl-d-erythritol-4-phospate (MEP) pathway was responsible for saponin accumulation in tea plants, and 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), diphosphomevalonate decarboxylase (MVD) and isopentenyl diphosphate isomerase (IDI) may be the key enzymes involved in saponin biosynthesis in tea seeds and flowers. Moreover, ten transcription factors (TFs) were predicted to regulate saponin biosynthesis in the tea plant. Taken together, our study provides a global insight into the saponin biosynthesis and accumulation in the tea plant.

Identification and characterization of SlbHLH, SlDof and SlWRKY transcription factors interacting with SlDPD gene involved in costunolide biosynthesis in Saussurea lappa

The genes involved in costunolide biosynthesis in Saussurea lappa have been identified recently by our lab. However, the study of transcriptional regulators of these genes was lacking for better opportunities for engineering the pharmacologically important biosynthetic pathway. Therefore, we cloned the promoter region of diphosphomevalonate decarboxylase gene (DPD) and analyzed its cis-acting regulatory elements to reveal the potential transcription factor (TF) binding sites for Dof, bHLH and WRKY family proteins in the gene promoter. The transcriptome study approach followed by the hidden Markov model based search, digital gene expression, co-expression network analysis, conserved domain properties and evolutionary analyses were carried out to screen out seven putative TFs for the DPD-TF interaction studies. Yeast one-hybrid assays were performed and three TFs were reported, namely, SlDOF2, SlbHLH3 and SlWRKY2 from Dof, bHLH and WRKY families, respectively that interacted positively with the DPD gene of the costunolide biosynthetic pathway. The tissue specific relative gene expression studies also supported the linked co-expression of the gene and its interacting TFs The present report will improve the understanding of transcriptional regulation pattern of costunolide biosynthetic pathway.

29 Influence of dietary manganese source on the corpus luteum proteome in pigs

Abstract Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine. Manganese (Mn) could be critical in regulating CL function since it is a cofactor for the enzyme Mn superoxide dismutase (Mn-SOD) as well as enzymes involved in progesterone (P4) synthesis. We hypothesized a more bioavailable dietary Mn source would increase CL Mn content thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Post-pubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded NRC requirements and was formulated to contain 20 ppm added Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2) or 60 (TRT3) ppm Mn from a Mn-amino acid complex (Availa-Mn; Zinpro Corporation) in place of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through D12 of the ensuing estrous cycle when gilts were euthanized. Mn content increased (P ≤ 0.06) 19, 21 and 24% in CLs of gilts fed TRT1, TRT2, and TRT3, respectively, and luteal P4 concentration decreased (P ≤ 0.03) 25, 26, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Total CL protein was extracted and liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed to assess global protein abundance. Compared to CON, 29, 105, and 118 proteins were differentially abundant (P < 0.01) in TRT1, TRT2, and TRT3, respectively. KEGG pathway analysis revealed proteins involved in P4 signaling (membrane-associated P4 receptor component 2) and cholesterol synthesis and transport (mevalonate kinase, diphosphomevalonate decarboxylase, low density lipoprotein receptor) were downregulated in response to Availa-Mn. Collectively, these data support the posit that dietary Mn source affects Mn accumulation and P4 concentration in CL tissue and influences protein abundance which may affect CL function.

Optimization of the IPP-bypass mevalonate pathway and fed-batch fermentation for the production of isoprenol in Escherichia coli

Isoprenol (3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals. Biological production of isoprenol via the mevalonate pathway has been developed and optimized extensively in Escherichia coli, but high ATP requirements and isopentenyl diphosphate (IPP) toxicity have made it difficult to achieve high titer, yield, and large-scale production. To overcome these limitations, an IPP-bypass pathway was previously developed using the promiscuous activity of diphosphomevalonate decarboxylase, and enabled the production of isoprenol at a comparable yield and titer to the original pathway. In this study, we optimized this pathway, substantially improving isoprenol production. A titer of 3.7 g/L (0.14 g isoprenol per g glucose) was achieved in batch conditions using minimal medium by pathway optimization, and a further optimization of the fed-batch fermentation process enabled an isoprenol titer of 10.8 g/L (yield of 0.105 g/g and maximum productivity of 0.157 g L-1 h-1), which is the highest reported titer for this compound. The substantial increase in isoprenol titer via the IPP-bypass pathway in this study will facilitate progress toward commercialization.

Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production.

Isoprene is a useful phytochemical with high commercial values in many industrial applications including synthetic rubber, elastomers, isoprenoid medicines, and fossil fuel. Currently, isoprene is on large scale produced from petrochemical sources. An efficient biological process for isoprene production utilizing renewable feedstocks would be an important direction of research due to the fossil raw material depletion and air pollution. In this study, we introduced the mevalonate (MVA) pathway genes/acetoacetyl-coenzyme A thiolase (mvaE) and MVA synthase (mvaS) from Enterococcus faecalis (E. faecalis); MVA kinase (mvk) derived from Methanosarcina mazei (M. mazei); and phosphomevalonate kinase (pmk), diphosphomevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (idi) from Streptococcus pneumoniae (S. pneumoniae) to accelerate dimethylallyl diphosphate (DMAPP) accumulation in Escherichia coli (E. coli). Together with a codon-optimized isoprene synthase (ispS) from Populus alba (P. alba), E. coli strain succeeded in formation of isoprene. We then manipulated the heterologous MVA pathway for high-level production of isoprene, by controlling the gene expression levels of the MVA pathway genes. We engineered four E. coli strains which showed different gene expression levels and different isoprene productivities, and we also characterized them with quantitative real-time PCR and metabolite analysis. To further improve the isoprene titers and release the toxicity to cells, we developed the extraction fermentation by adding dodecane in cultures. Finally, strain BL2T7P1TrcP harboring balanced gene expression system produced 587 ± 47mg/L isoprene, with a 5.2-fold titer improvement in comparison with strain BL7CT7P. This work indicated that a balanced metabolic flux played a significant role to improve the isoprene production via MVA pathway.

Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphate

The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon, Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate, trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract from A. pernix These data distinguish the modified mevalonate pathway of A. pernix and likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered from Thermoplasma acidophilum.

Enhanced Isoprene Production by Reconstruction of Metabolic Balance between Strengthened Precursor Supply and Improved Isoprene Synthase in Saccharomyces cerevisiae.

Isoprene, as a versatile bulk chemical, has wide industrial applications. Here, we attempted to improve isoprene biosynthesis in Saccharomyces cerevisiae by simultaneous strengthening of precursor supply and conversion via a combination of pathway compartmentation and protein engineering. At first, a superior isoprene synthase mutant ISPSLN was created by saturation mutagenesis, leading to almost 4-fold improvement in isoprene production. Subsequent introduction of ISPSLN to strains with strengthened precursor supply in either cytoplasm or mitochondria implied an imperfect match between the synthesis and conversion of the isopentenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMAPP) pool. To reconstruct metabolic balance between the upstream and downstream flux, additional copies of diphosphomevalonate decarboxylase gene ( MVD1) and isopentenyl-diphosphate delta-isomerase gene ( IDI1) were introduced into the cytoplasmic and mitochondrial engineered strains. Finally, the diploid strain created by mating the above haploid strains produced 11.9 g/L of isoprene, the highest ever reported in eukaryotic cells.

Molecular cloning, expression and immunolocalization analysis of diphosphomevalonate decarboxylase involved in terpenoid biosynthesis from Euphorbia helioscopia L.

ABSTRACTEuphorbia helioscopia L. is an herbaceous species of Euphorbia (Euphorbiaceae). As an ancient folk herbal medicine, the most effective medicinal component is terpenoid. Diphosphomevalonate decarboxylase (MDC) is the last rate-limiting enzyme of generating the isopentenyl pyrophosphate precursor of terpenoid in MVA pathway. The gene of MDC was cloned successfully from E. helioscopia, and named EhMDC (accession number: KP995936). The full length cDNA of EhMDC was 1653 bp, and it contained an open reading frame of 1245 bp, a 5′ untranslated region of 184 bp and a 3′ untranslated region of 224 bp. EhMDC contained 415 amino acids. Homologous sequence analysis showed that amino acid sequence of EhMDC had the highest identity of 88% with Ricinus communis MDC. Phylogenetic analysis of EhMDC indicated E. helioscopia, Hevea brasiliensis, R. communis, and Jatropha carcas which all belonged to Euphorbiaceae were classified into one class. Real-time PCR assay demonstrated EhMDC was constitutively expressed in roots, stems and leaves with a similar transcription level. Furthermore, in combination with immunoblot analysis and transmission electron microscopy immunogold labeling after anti-EhMDC antibody preparation, the EhMDC was found to be located in the cisternae of the endoplasmic reticulum, small vacuoles from endoplasmic reticulum and cytoplasm of laticifers. As a result, terpenoid biosynthesis site and accumulation of E. helioscopia laticifers were speculated.

A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.

Effect of copper nanoparticles exposure in the physiology of the common carp (Cyprinus carpio): Biochemical, histological and proteomic approaches

Copper nanoparticles (Cu-NPs) are serious water pollutants but their impact in teleosts performance remains poorly understood. In the present study, we have exposed juvenile carps (Cyprinus carpio), a freshwater teleost edible in India to two different doses (20 and 100 μg/L) of Cu-NPs for seven days. The doses selected were eco-relevant considering the contamination levels of certain water resources. The results indicated that the activity oxidative stress enzymes catalase, superoxide dismutase, and glutathione-S-transferase were significantly increased in the kidney, liver and gills of the treated groups when compared to control. Histological analysis revealed that after exposure, disruption of the secondary lamellae of gills, liver damage with pyknotic nuclei and structural disarray of the kidney occurred. Proteomic analysis of the liver showed down-regulation of several proteins including the ferritin heavy chain, rho guanine nucleotide exchange factor 17-like, cytoglobin-1 and up-regulation of diphosphomevalonate decarboxylase and selenide & water dikinase-1. Taken together, the results of suggest that short-term exposure of juvenile carp to Cu-NPs causes oxidative stress and impart serious deleterious effects in the tissues which may affect fish growth and development.

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus.

In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of β-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. This study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.

Development of a high-density SNP-based linkage map and detection of yellow pigment content QTLs in durum wheat

High-density genetic linkage maps of crop species are particularly useful in detecting qualitative and quantitative trait loci (QTLs) for agronomically important traits and in improving the power of classical approaches to identify candidate genes. The aim of this study was to develop a high-density genetic linkage map in a durum wheat recombinant inbred lines population derived from two elite wheat cultivars and to identify and characterize QTLs for yellow pigment content (YPC). A dense map was constructed by genotyping with the wheat 90K iSelect array and consisted of 5,670 loci, comprising 5,019 single nucleotide polymorphism (SNP), 467 DArT, 182 SSR markers and eight genes distributed in 35 linkage groups. Data for yellow pigment content were obtained from four replicated trials conducted at two locations in southern Italy for 2 years. A total of seven QTLs on different chromosome regions (1B, 2A, 2B, 5A, 5B, 7A and 7B) were identified, three of which were consistent in three or four environments and across environments. The genome scan for QTLs and the SNP homology prediction against annotated proteins in wheat and Brachypodium genomes identified two candidate genes of the carotenoid biosynthesis pathway (aldehyde oxidase, AO1, and diphosphomevalonate decarboxylase, DMAPD) significantly associated with YPC. This study provides a basis for further genetic QTL dissection and tools for marker-assisted breeding programs, because SNP markers and some carotenoid candidate genes were found to be tightly linked to major QTLs for YPC.

(R)-Mevalonate 3-Phosphate Is an Intermediate of the Mevalonate Pathway in Thermoplasma acidophilum

The lack of a few conserved enzymes in the classical mevalonate pathway and the widespread existence of isopentenyl phosphate kinase suggest the presence of a partly modified mevalonate pathway in most archaea and in some bacteria. In the pathway, (R)-mevalonate 5-phosphate is thought to be metabolized to isopentenyl diphosphate via isopentenyl phosphate. The long anticipated enzyme that catalyzes the reaction from (R)-mevalonate 5-phosphate to isopentenyl phosphate was recently identified in a Cloroflexi bacterium, Roseiflexus castenholzii, and in a halophilic archaeon, Haloferax volcanii. However, our trial to convert the intermediates of the classical and modified mevalonate pathways into isopentenyl diphosphate using cell-free extract from a thermophilic archaeon Thermoplasma acidophilum implied that the branch point intermediate of these known pathways, i.e. (R)-mevalonate 5-phosphate, is unlikely to be the precursor of isoprenoid. Through the process of characterizing the recombinant homologs of mevalonate pathway-related enzymes from the archaeon, a distant homolog of diphosphomevalonate decarboxylase was found to catalyze the phosphorylation of (R)-mevalonate to yield (R)-mevalonate 3-phosphate. The product could be converted into isopentenyl phosphate, probably through (R)-mevalonate 3,5-bisphosphate, by the action of unidentified T. acidophilum enzymes fractionated by anion-exchange chromatography. These findings demonstrate the presence of a third alternative "Thermoplasma-type" mevalonate pathway, which involves (R)-mevalonate 3-phosphotransferase and probably both (R)-mevalonate 3-phosphate 5-phosphotransferase and (R)-mevalonate 3,5-bisphosphate decarboxylase, in addition to isopentenyl phosphate kinase.

Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.