4-hydroxybenzoate Polyprenyltransferase Research Articles

Nitric oxide is involved in regulating cytochrome P450 gene family and mitochondrial related genes of Pleurotus eryngii in response to cadmium stress: preliminary expression patterns analysis

ABSTRACT   Nitric oxide (NO) plays an important role in fungal response to abiotic stress, but little is known about whether or not NO is involved in regulating cytochrome P450 (CYP) gene family and mitochondrial related genes (MRG) of Pleurotus eryngii in response to cadmium (Cd) stress. In this study, 125 CYP members and 127 differentially expressed MRG were identified. The analysis of gene length and exon number revealed the complexity of CYP gene family. After adding exogenous NO, the expression patterns showed that PleCYP2 and 4-hydroxybenzoate polyprenyltransferase were significantly up-regulated, the functions of membrane and membrane components were enhanced, and the mitochondrial splicing apparatus component gene was activated, which might be the potential mechanism of NO regulating P. eryngii resistance to Cd stress. This study lays a foundation for further revealing the mechanism of NO in regulating P. eryngii response to abiotic stress and promotes the potential application in fungal cultivation.

Plasmodium falciparum COQ2 gene encodes a functional 4-hydroxybenzoate polyprenyltransferase.

Ubiquinone (UQ) is a fundamental mitochondrial electron transport chain component. This compound is synthesized as the condensation of a p-substituted benzoic acid and a polyisoprenic moiety catalyzed by the enzyme 4-hydroxybenzoate polyprenyltransferase (EC 2.5.1.39). In Plasmodium spp., this enzyme is still uncharacterized. In this work, we expressed the sequence of the Plasmodium falciparum PF3D7_0607500 gene (abbreviated as PfCOQ2) in a coq2Δ mutant strain of Saccharomyces cerevisiae, and studied the functionality of its gene product. This open reading frame could complement S. cerevisiae coq2Δ mutant growth defect on media with glycerol as a carbon source. Further, UQ was unequivocally identified in lipid extracts from this coq2Δ mutant when expressing PfCOQ2. Remarkably, UQ was detected under those conditions when S. cerevisiae cells were metabolically labeled with either [ring-14C(U)]-p-aminobenzoic acid or [ring-14C(U)]-4-hydroxybenzoic acid. However, no UQ was detected in P. falciparum if labeled with p-aminobenzoic acid. These results indicate that PfCOQ2 is a 4-hydroxybenzoate polyprenyltransferase. Further, its substrate profile seems not dissimilar to that of S. cerevisiae, but, as in other organisms, p-aminobenzoic acid does not act as an aromatic precursor in UQ biosynthesis in P. falciparum. The reason for this last feature remains to be established, but may lie upstream of PfCOQ2.

Enhancement of antroquinonol production via the overexpression of 4-hydroxybenzoate polyprenyltransferase biosynthesis-related genes in Antrodia cinnamomea

Antroquinonol (AQ) as one of the most potent bioactive components in Antrodia cinnamomea (Fomitopsidaceae) shows a broad spectrum of anticancer effects. The lower yield of AQ has hampered its possible clinical application. AQ production may potentially be improved by genetic engineering. In this study, the protoplast-polyethylene glycol method combined with hygromycin as a selection marker was used in the genetic engineering of A. cinnamomea S-29. The optimization of several crucial parameters revealed that the optimal condition for generating maximal viable protoplasts was digestion of 4-day-old germlings with a mixture of enzymes (lysing enzyme, snailase, and cellulase) and 1.0 M MgSO4 for 4 h. The ubiA and CoQ2 genes, which are involved in the synthesis of 4-hydroxybenzoate polyprenyltransferase, were cloned and overexpressed in A. cinnamomea. The results showed that ubiA and CoQ2 overexpression significantly increased AQ production in submerged fermentation. The overexpressing strain produced maximum AQ concentrations of 14.75 ± 0.41 mg/L and 19.25 ± 0.29 mg/L in pCT74-gpd-ubiA and pCT74-gpd-CoQ2 transformants, respectively. These concentrations were 2.00 and 2.61 times greater than those produced by the control, respectively. This research exemplifies how the production of metabolites may be increased by genetic manipulation, and will be invaluable to guide the genetic engineering of other mushrooms that produce medically useful compounds.

Comparative Analysis of the Characteristics of Triterpenoid Transcriptome from Different Strains of Wolfiporia cocos.

The dried sclerotia of Wolfiporia cocos (Schwein.) Ryvarden & Gilb., a traditional Chinese medicine, has triterpenoid as its main active component. Breeding high-yield triterpenoid in W. cocos is an important research topic at present. We screened out two monosporal strains from the same W. cocos 5.78, high-yielding DZAC-Wp-H-29 (H) and low-yielding DZAC-Wp-L-123 (L), and cultured mycelia for 17 days, 34 days, and 51 days, respectively. Transcriptome analysis results showed that triterpenoid synthesis is closely related to gene expression in triterpenoid synthesis pathways (hydroxymethyl glutaryl-CoA reductase (HMGCR), farnesyl diphosphate synthase (FDPS), 4-hydroxybenzoate polyprenyltransferase (COQ2), C-8 sterol isomerase (ERG2), sterol O-acyltransferase (ACAT), tyrosine aminotransferase (TAT), torulene dioxygenase (CAO2), and sterol-4alpha-carboxylate 3-dehydrogenase (erg26)), and is limited by the expression of enzyme M20 combined with domain protein peptide (Pm20d2), aryl-alcohol dehydrogenase (norA), ISWI chromatin-remodeling complex ATPase ISW2, GroES-like protein (adh), cytochrome P450 (ftmP450-1), and unknown proteins unigene0001029 and unigene0011374. In addition, maintaining high triterpenoid accumulation in W. cocos may require a stable membrane structure, so the accumulation ability may be related to the high synthesis ability of sterols. The low accumulation of triterpenoid in W. cocos may be due to the products of key enzymes increasing flow to other pathways.

Genetic manipulation of the bifunctional gene, carRA, to enhance lycopene content in Blakeslea trispora

Blakeslea trispora is an ideal natural source of lycopene. Here, we genetically manipulated the bifunctional protein gene, carRA, encoding a phytoene synthase and lycopene cyclase, to enhance lycopene production. carR- and carRA-knockout and carA overexpressing strains were obtained after several rounds of selection. Following combinational fermentation for 6 d, the lycopene content in carR-knockout strains increased from 0.47 to 1.40mg/gDW, while the total carotenoid content decreased from 17.05 to 3.52mg/gDW compared to the wild type. No carotenoid was produced in carRA-knockout strains. In carA overexpressing strains, lycopene content, β-carotene, and total carotenoid content increased 2.26-, 1.98-, and 2.02-fold, resepectively, with proportions similar to wild type. Dry weight increased 1.89–1.98-fold in carR- and carRA-knockout strains compared to the wild type, but not in carA overexpressing strains. qRT-PCR analysis revealed that disruption of CarR and CarRA down-regulated carotenoid synthesis genes, especially the isopentenyl pyrophosphate isomerase gene, whereas overexpression of carA up-regulated all examined genes, except the ergosterol synthase and the 4-hydroxybenzoate polyprenyl transferase genes. Tripropylamine was shown to target the R domain, rather than the A domain, of CarRA. Furthermore, the possibility of using a carR-knockout to obtain high-lycopene B. trispora strains for industrial production was explored.

New susceptible variant of COQ2 gene in Japanese patients with sporadic multiple system atrophy.

Objective:The aim of this study was to analyze the association between the variations of coenzyme Q2 4-hydroxybenzoate polyprenyltransferase gene (COQ2) and Japanese patients with multiple system atrophy (MSA).Methods:We investigated the genetic variations in exons 1, 2, 6, and 7 of the COQ2 gene in 133 Japanese patients with MSA and 200 controls and analyzed the association between the variations and MSA.Results:Six DNA variations (G21S, L25V, V66L, P157S, V393A, and X422K) were found in the 133 patients with MSA, and G21S and X422K were new variations that had never been reported. V66L was a common variation that was found in all 133 patients with MSA. G21S, P157S, V393A, and X422K did not show gene frequency differences between patients with MSA and controls. On the other hand, L25V was newly proven to be the only risk factor of sporadic MSA with predominant olivopontocerebellar ataxia.Conclusions:The present study suggests L25V variant of COQ2 gene as a genetic risk factor in Japanese patients with MSA with cerebellar ataxia.

Mutation scanning of the COQ2 gene in ethnic Chinese patients with multiple-system atrophy

Multiple-system atrophy (MSA) is a fatal neurodegenerative disorder with unknown etiology. It is widely considered to be a nongenetic disorder, but accumulating evidence suggests that several genes are linked to MSA. Recently, functionally impaired variants in the coenzyme Q2 4-hydroxybenzoate polyprenyltransferase (COQ2) gene have been reported to increase the risk of MSA in familial and sporadic Japanese patients. In this study, we investigated the mutation spectrum of COQ2 and analyzed the association between the common variant Val393Ala in exon 7 of COQ2 and MSA in a Chinese population. This study included 312 sporadic MSA patients from the Department of Neurology, West China Hospital of Sichuan University. All 7 exons of COQ2 in all the patients and exon 7 in 598 healthy controls (HCs) were directly sequenced. Novel candidate mutations and variations were confirmed by direct sequencing in 300 HCs. Two novel nonsynonymous variants, including p.R173H and p.N386I, and a reported missense variant, p.L162F, were found in 4 patients (p.R173H in 2 patients). However, the Val393Ala variant was not detected in the above 4 patients. Thirteen MSA patients (4.17%) and 18 controls (3.01%) had the heterozygous variant (Val393Ala/NM) of COQ2. No significant differences existed in the genotype frequency and minor allele frequency of Val393Ala between patients and controls or between MSA characterized predominantly by cerebellar ataxia and by pakinsonism groups. The mutation frequency of COQ2 is 1.28% in a Chinese MSA population. The common variant Val393Ala in COQ2 does not appear to be associated with MSA in ethnic Chinese.

Identification and Active Expression of the Mycobacterium tuberculosis Gene Encoding 5-Phospho-α-D-ribose-1-diphosphate: Decaprenyl-phosphate 5-Phosphoribosyltransferase, the First Enzyme Committed to Decaprenylphosphoryl-D-arabinose Synthesis

Decaprenylphosphoryl-d-arabinose, the lipid donor of mycobacterial d-arabinofuranosyl residues, is synthesized from phosphoribose diphosphate rather than from a sugar nucleotide. The first committed step in the process is the transfer of a 5-phosphoribosyl residue from phosphoribose diphosphate to decaprenyl phosphate to form decaprenylphosphoryl-5-phosphoribose via a 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phospho-ribosyltransferase. A candidate for the gene encoding this enzyme (Rv3806c) was identified in Mycobacterium tuberculosis, primarily via its homology to one of four genes responsible for d-arabinosylation of nodulation factor in Azorhizobium caulinodans. The resulting protein was predicted to contain eight or nine transmembrane domains. The gene was expressed in Escherichia coli, and membranes from the expression strain of E. coli but not from a control strain of E. coli were shown to convert phosphoribose diphosphate and decaprenyl phosphate into decaprenylphosphoryl-5-phosphoribose. Neither UDP-galactose nor GDP-mannose was active as a sugar donor. The enzyme favored polyprenyl phosphate with 50-60 carbon atoms, was unable to use C-20 polyprenyl phosphate, and used C-75 polyprenyl phosphate less efficiently than C-50 or C-60. It requires CHAPS detergent and Mg(2+) for activity. The Rv3806c gene encoding 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase is known to be essential for the growth of M. tuberculosis, and the tuberculosis drug ethambutol inhibits other steps in arabinan biosynthesis. Thus the Rv3806c-encoded enzyme appears to be a good target for the development of new tuberculosis drugs.

Characterization of Polyprenyldiphosphate: 4-Hydroxybenzoate Polyprenyltransferase from Escherichia coli

Polyprenyldiphosphate: 4-hydroxybenzoate polyprenyltransferase (4-HB polyprenyltransferase) is a key enzyme in ubiquinone biosynthesis in E. coli, encoded by the gene ubiA. By overexpression of ubiA and isolation of the membrane fraction, the enzyme was enriched approx. 3000-fold and characterized. The enzyme is membrane-bound and could not be solubilized by hypotonic buffer or detergent treatment. The enzymatic activity is optimal at pH 7.8 and depends on the presence of magnesium ions. Geranyldiphosphate (GPP), all- trans-farnesyldiphosphate (FPP) and all- trans-solanesyldiphosphate (SPP) are accepted as side chain precursors. The apparent K m values for these substances are are 254 μM, 22 μM and 31 μM, respectively. No reaction was observed with ω-t 2-c 5-octaprenyldiphosphate, in which five double bounds have cis-configuration. The reaction is stimulated by 0.01% CHAPS, but strongly inhibited by sodiumdeoxycholate, Tween 80 and Triton X-100. The amino acid sequence shows striking similarities to 4-HB hexaprenyltransferase from yeast. Sequence homologies to other prenyltransferases are discussed.

The 92-min region of the Escherichia coli chromosome: location and cloning of the ubi and alr

A cosmid (pND320) bearing 42.5 kb of Escherichia coli chromosomal DNA, including the genes between xylE and ssb near minute 92 on the linkage map, was isolated by selection for complementation of a dnaB mutation. Known nucleotide (nt) sequences were used to align restriction maps in this region to the physical map of the chromosome (coordinates 4319.5 to 4362 kb), and to locate precisely and define the orientations of 19 genes. Predicted physical linkage of sequenced genes across unsequenced gaps of defined length was confirmed by the nt sequence analysis of fragments subcloned from pND320. Mutant complementation by plasmids showed that ubiA is located between malM and plsB. A previously sequenced long open reading frame that encodes the C-terminal portion of the E. coli ubiA product (4-hydroxybenzoate polyprenyltransferase, HPTase) shows a high degree of sequence identity with the corresponding segment of yeast HPTase (the COQ2 gene product). Comparison of homologous regions from E. coli and Salmonella typhimurium was used to locate precisely the gene alr that encodes alanine racemase (ARase) between dnaB and tyrB. Subcloning of alr downstream from tandem bacteriophage lambda promoters produced a plasmid that directed high-level overproduction of a soluble approx. 40-kDa protein with ARase activity.

[37] 4-Hydroxybenzoate polyprenyltransferase from rat liver

This chapter focuses on 4-hydroxybenzoate polyprenyltransferase from rat liver. 4-Hydroxybenzoate polyprenyltransferase is a key enzyme in ubiquinone synthesis because it catalyzes the step that brings together the precursor of the benzoquinone ring and the polyisoprenoid side chain. The substrates are 4-hydroxybenzoate derived from tyrosine metabolism, and polyprenyl pyrophosphates derived from mevalonic acid and the product is 3-polyprenyl-4-hydroxybenzoate (PPHB) and pyrophosphate. The 3-polyprenyl-4-hydroxybenzoate, after hydroxylation, decarboxylation, and methylation, gives rise to ubiquinone. When nonaprenyl pyrophosphate (NPP) was used as a source of polyprenyl side chain in rat liver, 3-nonaprenyl-4-hydroxybenzoate was the major product but there was also minor incorporation into 3-octaprenyl-4 hydroxybenzoate. The formation of octaprenyl derivative might be due to observed contamination of solanesol, which was used for the preparation of nonaprenyl pyrophosphate. In rat liver, the addition of IPP along with NPP resulted in formation of a minor amount of decaprenyl derivative but still nonaprenyl derivative was the major component of the products formed. Whereas in guinea pig liver, the addition of IPP along with NPP resulted in formation of octa, nona, and decaprenyl derivatives all in significant quantities.