The hydroxyphenylpyruvate dioxygenase from Synechocystis sp. PCC 6803 is not required for plastoquinone biosynthesis

Recently it has been reported that macrophages express a nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ). Using a ligand of PPARγ, troglitazone or pioglitazone, we have shown that the expression of two genes involved in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase, were increased by activation of PPARγ through a PPAR response element (PPRE) in THP-1 macrophages. In addition, treatment with troglitazone significantly increased the activity of HMG-CoA reductase and the amount of intracellular cholesterol. Thus, we conclude that PPARγ and its agonists increase the cholesterol content of macrophages by the increased expression of genes involved in cholesterol biosynthesis. These findings suggest that PPARγ may play a role in cholesterol metabolism in macrophages.     

Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.     

Hormonal Regulation of the mRNA Encoding the Ketogenic Enzyme Mitochondrial 3-Hydroxy-3-Methylglutaryl-CoA Synthase in Neonatal Primary Cultures of Cortical Astrocytes and Meningeal Fibroblasts

Abstract: We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3-hydroxy-3-methylglutaryl-CoA cycle, namely, mitochondrial acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum-free/hormone-free medium.     

高产辅酶Q10结构类似物抗性突变株的选育

以土壤杆菌(Agrobacteriumsp.)TLY-4为出发菌株,采用70%致死剂量的NTG进行诱变处理,通过筛选抗辅酶Q10结构类似物维生素K3突变株,定向选育到了两株辅酶Q10高产突变株,编号为R-122和R-015,其摇瓶发酵72h时的辅酶Q10产量分别为57.3 mg/L和59.9 mg/L,较出发菌株提高了35.7%和41.6%。通过连续传代实验,表明突变株高产辅酶Q10的遗传性状稳定。实验以有机溶剂DMF和吐温-80共同增溶的方法,解决了维生素K3在培养基中易析出的问题,并确定了平板培养基中维生素K3的最小抑菌浓度为0.15 mg/mL。     

Biosynthesis and physiology of coenzyme Q in bacteria

Ubiquinone, also called coenzyme Q, is a lipid subject to oxido-reduction cycles. It functions in the respiratory electron transport chain and plays a pivotal role in energy generating processes. In this review, we focus on the biosynthetic pathway and physiological role of ubiquinone in bacteria. We present the studies which, within a period of five decades, led to the identification and characterization of the genes named ubi and involved in ubiquinone production in Escherichia coli. When available, the structures of the corresponding enzymes are shown and their biological function is detailed. The phenotypes observed in mutants deficient in ubiquinone biosynthesis are presented, either in model bacteria or in pathogens. A particular attention is given to the role of ubiquinone in respiration, modulation of two-component activity and bacterial virulence. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Properties and tissue distribution of a novel aldo-keto reductase encoding in a rat gene (Akr1b10)

A recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence similarity to human aldo-keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10 (R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 utilized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and α-dicarbonyl compounds (such as methylglyoxal and 3-deoxyglucosone), showing low K_m values of 0.8-6.1 μM and 3.7-67 μM, respectively. The enzyme also reduced glyceraldehyde and tetroses (K_m = 96-390 μM), although hexoses and pentoses were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most efficiently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress and glycation in rat tissues.     

An evolutionarily conserved alternate metal ligand is important for activity in α-isopropylmalate synthase from Mycobacterium tuberculosis

Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon–carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes.     

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.     

The idebenone metabolite QS10 restores electron transfer in complex I and coenzyme Q defects

Idebenone is a hydrophilic short-chain coenzyme (Co) Q analogue, which has been used as a potential bypass of defective complex I in both Leber Hereditary Optic Neuropathy and OPA1-dependent Dominant Optic Atrophy. Based on its potential antioxidant effects, it has also been tested in degenerative disorders such as Friedreich's ataxia, Huntington's and Alzheimer's diseases. Idebenone is rapidly modified but the biological effects of its metabolites have been characterized only partially. Here we have studied the effects of quinones generated during in vivo metabolism of idebenone with specific emphasis on 6-(9-carboxynonyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (QS10). QS10 partially restored respiration in cells deficient of complex I or of CoQ without inducing the mitochondrial permeability transition, a detrimental effect of idebenone that may offset its potential benefits [Giorgio et al. (2012) Biochim. Biophys. Acta 1817: 363–369]. Remarkably, respiration was largely rotenone-insensitive in complex I deficient cells and rotenone-sensitive in CoQ deficient cells. These findings indicate that, like idebenone, QS10 can provide a bypass to defective complex I; and that, unlike idebenone, QS10 can partially replace endogenous CoQ. In zebrafish (Danio rerio) treated with rotenone, QS10 was more effective than idebenone in allowing partial recovery of respiration (to 40% and 20% of the basal respiration of untreated embryos, respectively) and allowing zebrafish survival (80% surviving embryos at 60?h post-fertilization, a time point at which all rotenone-treated embryos otherwise died). We conclude that QS10 is potentially more active than idebenone in the treatment of diseases caused by complex I defects, and that it could also be used in CoQ deficiencies of genetic and acquired origin.     

Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.     

Intracellular cholesterol accumulation and coenzyme Q10 deficiency in Familial Hypercholesterolemia

Familial Hypercholesterolemia (FH) is an autosomal co-dominant genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature cardiovascular disease. Here, we examined FH pathophysiology in skin fibroblasts derived from FH patients harboring heterozygous mutations in the LDL-receptor.Fibroblasts from FH patients showed a reduced LDL-uptake associated with increased intracellular cholesterol levels and coenzyme Q₁₀ (CoQ₁₀) deficiency, suggesting dysregulation of the mevalonate pathway.Secondary CoQ₁₀ deficiency was associated with mitochondrial depolarization and mitophagy activation in FH fibroblasts. Persistent mitophagy altered autophagy flux and induced inflammasome activation accompanied by increased production of cytokines by mutant cells. All the pathological alterations in FH fibroblasts were also reproduced in a human endothelial cell line by LDL-receptor gene silencing.Both increased intracellular cholesterol and mitochondrial dysfunction in FH fibroblasts were partially restored by CoQ₁₀ supplementation. Dysregulated mevalonate pathway in FH, including increased expression of cholesterogenic enzymes and decreased expression of CoQ₁₀ biosynthetic enzymes, was also corrected by CoQ₁₀ treatment.Reduced CoQ₁₀ content and mitochondrial dysfunction may play an important role in the pathophysiology of early atherosclerosis in FH. The diagnosis of CoQ₁₀ deficiency and mitochondrial impairment in FH patients may also be important to establish early treatment with CoQ₁₀.     

Multi-target-directed design,syntheses, and characterization of fluorescent bisphosphonate derivatives as multifunctional enzyme inhibitors in mevalonate pathway

Background

Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. Four enzymes in mevalonate pathway, including MVK, PMK, MDD, and FPPS, play important regulatory roles in cholesterol biosynthesis and cell proliferation.

Methods

The following methods were used: cloning, expression and purification of enzymes in mevalonate pathway, organic syntheses of multifunctional enzyme inhibitors, measurement of their IC₅₀ values for above four enzymes, kinetic studies of enzyme inhibitions, molecular modeling studies, cell viability tests, and fluorescence microscopy.

Results and conclusions

We report our multi-target-directed design, syntheses, and characterization of two blue fluorescent bisphosphonate derivatives compounds 15 and 16 as multifunctional enzyme inhibitors in mevalonate pathway. These two compounds had good inhibition to all these four enzymes with their IC₅₀ values at nanomolar to micromolar range. Kinetic and molecular modeling studies showed that these two compounds could bind to the active sites of all these four enzymes. The fluorescence microscopy indicated that these two compounds could easily get into cancer cells.

General significance

Multifunctional enzyme inhibitors are generally more effective than single enzyme inhibitors, with fewer side effects. Our results showed that these multifunctional inhibitors could become lead compounds for further development for the treatment of soft-tissue tumors and hypercholesteremia.     

Cloning and expression analysis of ten genes associated with picrosides biosynthesis in Picrorhiza kurrooa

Picrorhiza kurrooa Royle ex Benth. is an economically important medicinal plant known to yield picrosides which have high medicinal value. Picroside I and picroside II are major picrosides associated with various bioactivities. The present work analyzed the expression of various genes of the picrosides biosynthesis pathway in different tissues of the plant in relation to the picrosides content. Eight full-length cDNA sequences namely, 1-deoxy-d-xylulose-5-phosphate synthase (2.317 kb), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (1.767 kb), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (1.674 kb), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (1.701 kb), acetyl-CoA acetyltransferase (1.545 kb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (2.241 kb), isopentenyl pyrophosphate isomerase (987 bp) and geranyl diphosphate synthase (1.434 kb), were cloned to full-length followed by expression analysis of ten genes vis-à-vis picrosides content analysis. There is maximum accumulation of picrosides in leaf tissue followed by the rhizome and root, and a similar pattern of expression was found in all the ten genes. The genes responded to the modulators of the picrosides biosynthesis. Picrosides accumulation was enhanced by application of hydrogen peroxide and abscisic acid, whereas methyl jasmonate and salicylic acid treatment decreased the content.     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.     

Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei

The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma.     

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.