Evolutionary recruitment of a flavin-dependent monooxygenase for stabilization of sequestered pyrrolizidine alkaloids in arctiids

Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants.     

Molecular cloning and characterization of a full-length cDNA clone for human plasminogen

A human liver cDNA library enriched for full-length clones was screened for plasminogen cDNA using a synthetic 24-nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra Ile at position 65, Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'-non-coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)-tail.     

Molecular cloning and catalytic mechanism of a novel glycosphingolipid-degrading beta-N-acetylgalactosaminidase from Paenibacillus sp. TS12

We report here the molecular cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading β-N-acetylgalactosaminidase (β-NGA) from Paenibacillus sp. TS12 (NgaP). Consisting of 1034 putative amino acid residues, NgaP shares no sequence similarity with known proteins. Recombinant NgaP, expressed in Escherichia coli, cleaved the nonreducing terminal β-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-β-N-acetylgalactosaminide ～100 times faster than para-nitrophenyl-β-N-acetylglucosaminide. GalNAc thiazoline, an analog of the oxazolinium intermediate and potent inhibitor for enzymes adopting substrate-assisted catalysis, competitively inhibited the enzyme. The K(i) of the enzyme for GalNAc thiazoline was 1.3 nM, whereas that for GlcNAc thiazoline was 46.8 μM. Comparison of the secondary structure with those of known enzymes exhibiting substrate-assisted catalysis and point mutation analysis indicated that NgaP adopts substrate-assisted catalysis in which Glu-608 and Asp-607 could function as a proton donor and a stabilizer of the 2-acetamide group of the β-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a β-NGA showing substrate-assisted catalysis. This is the first report describing the molecular cloning of a β-NGA adopting substrate-assisted catalysis.     

Identification, cloning, and characterization of two N-acetylgalactosamine-binding lectins from the albumen gland of Helix pomatia

Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA, is required. A combination of isoelectric focusing, amino acid sequence analysis, and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII), both consistent with GalNAc-binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity whereas molecular modeling using SWISS-MODEL suggests they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-thioredoxin-tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes whereas their oligosaccharide specificity, evaluated using glycan microarrays, showed that they predominantly bind glycans with terminal α-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (K(d) = 0.60 nm and 2.00 nm, respectively) with a somewhat higher affinity to native HPA (K(d) = 7.67 nm). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a manner similar to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.     

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.     

Molecular cloning and characterization of the polyphenol oxidase gene from sweetpotato

Polyphenol oxidase is the enzyme responsible for enzymatic browning in sweetpotato that decreases the commercial value of sweetpotato products. Here we reported the cloning and characterization of a new cDNA encoding PPO from sweetpotato, designated as IbPPO (GeneBank accession number: AY822711). The full-length cDNA of IbPPO is 1984 bp with a 1767 bp open reading frame (ORF) encoding a 588 amino acid polypeptide with a calculated molecular weight of 65.7 kDa and theoretical pI of 6.28. The coding sequence of IbPPO was also directly amplified from the genomic DNA of sweetpotato that demonstrated that IbPPO was an intron-free gene. The computational comparative analysis revealed that IbPPO showed homology to other PPOs of plant origin and contained a 50 amino acid plastidial transit peptide at its N-terminal and the two conserved CuA and CuB copper-binding motifs in the catalytic region of IbPPO. A highly conserved serine-rich motif was firstly found in the transit peptides of plant PPO enzymes. Then the homology based structural modeling of IbPPO showed that IbPPO had the typical structure of PPO: the catalytic copper center was accommodated in a central four-helix bundle located in a hydrophobic pocket close to the surface. Finally, the results of the semiquantitative RT-PCR analysis of IbPPO in different tissues demonstrated that IbPPO could express in all the organs of sweetpotato including mature leaves, young leaves, the stems of mature leaves (petioles), the storage roots, and the veins but at different levels. The highest-level expression of IbPPO was found in the veins, followed by storage roots, young leaves and mature leaves; and the lowest-level expression of IbPPO was found in petioles. The present researches will facilitate the development of antibrown sweetpotato by genetic engineering. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 6, pp. 1006–1012. The article was submitted by the authors in English.     

Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat.

Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha. The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.     

Molecular cloning and functional expression of a novel extracellular lipase from the thermotolerant yeast Candida thermophila

The thermotolerant yeast Candida thermophila SRY-09 isolated from Thailand produces an extracellular lipase that hydrolyses various triglycerides. To clone the gene encoding the lipase, Saccharomyces cerevisiae was transformed with a C. thermophila genomic library and screened for lipase activity on medium containing olive oil emulsion and rhodamine B. One C. thermophila lipase gene (CtLIP) was found that contained an ORF of 1317 bp encoding a deduced polypeptide of 438 amino acids. Candida thermophila lipase contained a Gly-Asp-Ser-Gln-Gly motif which matched the consensus Gly-X-Ser-X-Gly conserved among lipolytic enzymes. Heterologous expression of the cloned CtLIP under the control of the alcohol oxidase gene (AOX1) promoter in the methylotrophic yeast Pichia pastoris, and enzymatic measurements confirmed the function of the respective protein as a lipase. The recombinant CtLIP could hydrolyse various substrates at high temperature (55 degrees C) with higher efficiency than at 37 or 45 degrees C and preferentially hydrolysed two-positional ester bonds. As with C. thermophila, the heterologously expressed lipase was secreted into the medium by Pichia pastoris.     

Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybean seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened with a peroxidase-specific probe. The probe was generated by 3′ rapid amplification of cDNA ends with soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme ligand). Positive clones were recovered by PCR using the degenerate peroxidase-specific primer and the vector primer T₇ flanking the cloning site. Four cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 1326, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature proteins of 303, 303, 292, and 292 amino acids, respectively. The four predicted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% amino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity. GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and active forms of the two denatured proteins were recovered after in vitro folding in a medium containing hemin, urea and Ca²⁺. GmEpa1 and GmEpa2 messages were detected in developing seed and root, while GmEpb1 and GmEpb2 messages were present in root, leaf, stem and seed pod. These cDNAs and cDNA-specific primers will allow investigations into peroxidase’s role in development, stress response and in other physiological processes.     

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A₂ (PLA₂s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA₂ inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA₂s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated αBaltMIP, showed it to be a glycoprotein with Mr of ∼24,000 for the monomeric subunit. CD spectra of the PLA₂/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of αBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization.     

Molecular cloning of aromatic degradative genes from Pseudomonas stutzeri

Abstract Using dialysed cell-free extracts of the purple non-sulphur bacterium Rhodomicrobium vannielii protein kinase activities capable of transferring the gamma phosphate group from gamma [³²P]ATP to a variety of polypeptides were detected. The optimum concentration of Mg²⁺ for protein kinase activity was about 20 mM and the phosphorylation of one polypeptide ( M _r 47 kDa) was inhibited by chlorpromazine, a calmodulin antagonist, and also by Ca²⁺. The activity of at least one of the protein kinases (or a phosphatase) was regulated by ribulose 1,5-bisphosphate.     

Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida

Earthworm fibrinolytic enzyme (EFE), a multi-com-ponent protease purified from some earthworm breeds,belongs to serine protease family with fibrinolytic activity[1]. It has been used in prevention and treatment of cardiacand cerebrovascular diseases in Ch…     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.