Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

Complexity of gene paralogues resolved in biosynthetic pathway of hepatoprotective iridoid glycosides in a medicinal herb, Picrorhiza kurroa through differential NGS transcriptomes.

Picrorhiza kurroa is a medicinal herb with diverse pharmacological applications due to the presence of iridoid glycosides, picroside-I (P-I), and picroside-II (P-II), among others. Any genetic improvement in this medicinal herb can only be undertaken if the biosynthetic pathway genes are correctly identified. Our previous studies have deciphered biosynthetic pathways for P-I and P-II, however, the occurrence of multiple copies of genes has been a stumbling block in their usage. Therefore, a methodological strategy was designed to identify and prioritize paralogues of pathway genes associated with contents of P-I and P-II. We used differential transcriptomes varying for P-I and P-II contents in different tissues of P. kurroa. All transcripts for a particular pathway gene were identified, clustered based on multiple sequence alignment to notify as a representative of the same gene (≥ 99% sequence identity) or a paralogue of the same gene. Further, individual paralogues were tested for their expression level via qRT-PCR in tissue-specific manner. In total 44 paralogues in 14 key genes have been identified out of which 19 gene paralogues showed the highest expression pattern via qRT-PCR. Overall analysis shortlisted 6 gene paralogues, PKHMGR3, PKPAL2, PKDXPS1, PK4CL2, PKG10H2 and PKIS2 that might be playing role in the biosynthesis of P-I and P-II, however, their functional analysis need to be further validated either through gene silencing or over-expression. The usefulness of this approach can be expanded to other non-model plant species for which transcriptome resources have been generated.

Heterologous Expression and Characterization of Flavinadenine Dinucleotide Synthetase from Candida famata for Flavin Adenine Dinucleotide Production.

BACKGROUND: Flavin adenine dinucleotide (FAD) is a redox-active coenzyme that regulates several important enzymatic reactions during metabolism. FAD is used in the medicinal and food industries and FAD supplements have been used to treat some inheritable diseases. FAD can be biosynthesized from flavin mononucleotide (FMN) and adenosine triphosphate (ATP), catalyzed by FAD synthetase (FADS). OBJECTIVE: The aim of this study was to heterologously express the gene encoding FADS from the flavinogenic yeast Candida famata (FADSCf) for biosynthesis of FAD. METHODS: The sequence encoding FADSCf was retrieved and heterologously expressed in Escherichia coli. The structure and enzymatic properties of recombinant FADSCf were characterized. RESULTS: FADSCf (279 amino acids) was successfully expressed in E. coli BL21 (DE3), with a theoretical molecular weight of 32299.79 Da and an isoelectric point of 6.09. Secondary structural analysis showed that the number of α-helices was 2-fold higher than the number of ß-sheets, indicating that the protein was highly hydrophilic. Under fixed ATP concentration, FADSCf had a Km of 0.04737±0.03158 mM and a Vmax of 3.271±0.79 µM/min/mg. Under fixed FMN concentration, FADSCf had a Km of 0.1214±0.07464 mM and a Vmax of 2.6695±0.3715 µM/min/mg. Enzymatic reactions in vitro showed that expressed FADSCf could form 80 mM of FAD per mg of enzyme after 21 hours under the following conditions: 0.5 mM FMN, 5 mM ATP and 10 mM Mg2+. CONCLUSION: Under optimized conditions (0.5 mM FMN, 5 mM ATP and 10 mM Mg2+), the production of FAD reached 80 mM per mg of FADSCf after a 21-hour reaction. Our results indicate that purified recombinant FADSCf can be used for the biosynthesis of FAD.

RNAi-mediated knock-down of the dopamine beta-hydroxylase gene changes growth of razor clams.

Dopamine beta-hydroxylase (DßH) plays an essential role in the synthesis of catecholamines (CA) in neuroendocrine networks. In the razor clam, Sinonovacula constricta a novel gene for DßH (ScDßH-α) was identified that belongs to the copper type II ascorbate-dependent monooxygenase family. Expression analysis revealed ScDßH-α gene transcripts were abundant in the liver and expressed throughout development. Knock-down of ScDßH-α in adult clams using siRNA caused a reduction in the growth rate compared to control clams. Reduced growth was associated with strong down-regulation of gene transcripts for the growth-related factors, platelet derived growth factors A (PDGF-A) (P < 0.001) 24 h after ScDßH-α knock-down, vascular endothelial growth factor (VEGF1) (P < 0.001) and platelet derived growth factor B (PDGF-B-2) (P < 0.001) 24 h and 48 h after ScDßH-α knock-down and transforming growth factor beta (TGF-ß1) (P < 0.001) 48 h and 72 h after ScDßH-α knock-down. Taken together the results suggest that the novel ScDßH-α gene through its role in CA synthesis is involved in growth regulation in the razor clam and possibly other bivalves.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents.

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs.

A potential role for hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) in teleost acid-base and ammonia regulation.

Increasing evidence suggests the involvement of hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) not only in cardiac and neural function, but also in more general physiological processes including acid-base and ammonia regulation. We have identified four different HCN paralogs/isoforms in the goldfish Carassius auratus (CaHCN1, CaHCN2b, CaHCN4a and CaHCN4b) as likely candidates to contribute to renal, branchial and intestinal acid-base and ammonia regulation in this teleost. Quantitative real-time PCR showed not only high mRNA abundance of all isoforms in heart and brain, but also detectable levels (particularly of CaHCN2b and CaHCN4b) in non-excitable tissues, including gills and kidneys. In response to an internal or external acid-base and/or ammonia disturbance caused by feeding or high environmental ammonia, respectively, we observed differential and tissue-specific changes in mRNA abundance of all isoforms except CaHCN4b. Furthermore, our data suggest that the functions of specific HCN channels are supplemented by certain Rhesus glycoprotein functions to help in the protection of tissues from elevated ammonia levels, or as potential direct routes for ammonia transport in gills, kidney, and gut. The present results indicate important individual roles for each HCN isoform in response to acid-base and ammonia disturbances.

Identification and Characterization of NPR1 and PR1 Homologs in Cymbidium orchids in Response to Multiple Hormones, Salinity and Viral Stresses.

The plant nonexpressor of pathogenesis-related 1 (NPR1) and pathogenesis-associated 1 (PR1) genes play fundamental roles in plant immunity response, as well as abiotic-stress tolerance. Nevertheless, comprehensive identification and characterization of NPR1 and PR1 homologs has not been conducted to date in Cymbidium orchids, a valuable industrial crop cultivated as ornamental and medicinal plants worldwide. Herein, three NPR1-like (referred to as CsNPR1-1, CsNPR1-2, and CsNPR1-3) and two PR1-like (CsPR1-1 and CsPR1-2) genes were genome-widely identified from Cymbidium orchids. Sequence and phylogenetic analysis revealed that CsNPR1-1 and CsNPR1-2 were grouped closest to NPR1 homologs in Zea mays (sharing 81.98% identity) and Phalaenopsis (64.14%), while CsNPR1-3 was classified into a distinct group with Oryza sativa NPR 3 (57.72%). CsPR1-1 and CsPR1-2 were both grouped closest to Phalaenopsis PR1 and other monocot plants. Expression profiling showed that CsNPR1 and CsPR1 were highly expressed in stem/pseudobulb and/or flower. Salicylic acid (SA) and hydrogen peroxide (H2O2) significantly up-regulated expressions of CsNPR1-2, CsPR1-1 and CsPR1-2, while CsNPR1-3, CsPR1-1 and CsPR1-2 were significantly up-regulated by abscisic acid (ABA) or salinity (NaCl) stress. In vitro transcripts of entire Cymbidium mosaic virus (CymMV) genomic RNA were successfully transfected into Cymbidium protoplasts, and the CymMV infection up-regulated the expression of CsNPR1-2, CsPR1-1 and CsPR1-2. Additionally, these genes were transiently expressed in Cymbidium protoplasts for subcellular localization analysis, and the presence of SA led to the nuclear translocation of the CsNPR1-2 protein, and the transient expression of CsNPR1-2 greatly enhanced the expression of CsPR1-1 and CsPR1-2. Collectively, the CsNPR1-2-mediated signaling pathway is SA-dependent, and confers to the defense against CymMV infection in Cymbidium orchids.

The structure of MP-4 from Mucuna pruriens at 2.22†Šresolution.

The structure of the MP-4 protein was previously determined at a resolution of 2.8 Å. Owing to the unavailability of gene-sequence information at the time, the side-chain assignment was carried out on the basis of a partial sequence available through Edman degradation, sequence homology to orthologs and electron density. The structure of MP-4 has now been determined at a higher resolution (2.22 Å) in another space group and all of the structural inferences that were presented in the previous report of the structure were validated. In addition, the present data allowed an improved assignment of side chains and enabled further analysis of the MP-4 structure, and the accuracy of the assignment was confirmed by the recently available gene sequence. The study reinforces the traditional concept that conservative interpretations of relatively low-resolution structures remain correct even with the availability of high-resolution data.

Establishing a System for Functional Characterization of Full-Length cDNAs of Camellia sinensis.

: Tea (Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea's recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.

Identification and characterization of type III polyketide synthase genes from culturable endophytes of ethnomedicinal plants.

Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including Arbus precatorius, Bacopa monnieri,Citrus aurantifolia and Datura metel to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60-99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from Fusarium incarnatum BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in E. coli Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 104 s-1 M-1 with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.

Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication.

BACKGROUND: The recently published complete mitogenome of the European lobster (Homarus gammarus) that was generated using long-range PCR exhibits unusual gene composition (missing nad2) and gene rearrangements among decapod crustaceans with strong implications in crustacean phylogenetics. Such atypical mitochondrial features will benefit greatly from validation with emerging long read sequencing technologies such as Oxford Nanopore that can more accurately identify structural variation. RESULTS: We re-sequenced the H. gammarus mitogenome on an Oxford Nanopore Minion flowcell and performed a long-read only assembly, generating a complete mitogenome assembly for H. gammarus. In contrast to previous reporting, we found an intact mitochondrial nad2 gene in the H. gammarus mitogenome and showed that its gene organization is broadly similar to that of the American lobster (H. americanus) except for the presence of a large tandemly duplicated region with evidence of pseudogenization in one of each duplicated protein-coding genes. CONCLUSIONS: Using the European lobster as an example, we demonstrate the value of Oxford Nanopore long read technology in resolving problematic mitogenome assemblies. The increasing accessibility of Oxford Nanopore technology will make it an attractive and useful tool for evolutionary biologists to verify new and existing unusual mitochondrial gene rearrangements recovered using first and second generation sequencing technologies, particularly those used to make phylogenetic inferences of evolutionary scenarios.

Bioactivities and genome insights of a thermotolerant antibiotics-producing Streptomyces sp. TM32 reveal its potentials for novel drug discovery.

A way to defeat antimicrobial resistance (AMR) crisis is to supply novel drugs to the pharmaceutical industry. This effort leads to a global call for seeking the beneficial microbes from underexplored habitats. To support this call, we isolated Streptomyces sp. TM32 from the rhizosphere soil of a medicinal plant, turmeric (Curcuma longa L.). TM32 exhibited strong antimicrobial activities against both human and plant pathogens, including an AMR pathogen, Staphylococcus haemolyticus MR-CoNS. Surprisingly, such antimicrobial results of TM32's autoclaved crude extract remained the same. Based on the genome data analysis, TM32 belongs to the same genomic species with Streptomyces sioyaensis DSM 40032T , supported by the relatively high-average nucleotide identity values (ANIb: 96.80% and OrthoANIu: 97.14%) and in silico DNA-DNA relatedness value of 75.40%. Importantly, the gene annotation analyses revealed that TM32's genome contains various genes encoding the biosynthesis of either known or unknown antibiotics and some metabolites involved in plant growth-promoting traits. However, bioactivities and genome data comparison of TM32 and DSM 40032T showed a set of apparent differences, for example, antimicrobial potentials, genome size, number, and occurrence of coding DNA sequences in the chromosomes. These findings suggest that TM32 is a new strain of S. sioyaensis and serves as an emerging source for further discovery of valuable and novel bioactive compounds.

Characterization of YY1 OPB Peptide for its Anticancer Activity.

BACKGROUND: The oncoprotein binding (OPB) domain of Yin Yang 1 (YY1) consists of 26 amino acids between G201 and S226, and is involved in YY1 interaction with multiple oncogene products, including MDM2, AKT, EZH2 and E1A. Through the OPB domain, YY1 promotes the oncogenic or proliferative regulation of these oncoproteins in cancer cells. We previously demonstrated that a peptide with the OPB sequence blocked YY1-AKT interaction and inhibited breast cancer cell proliferation. OBJECTIVE: In the current study, we characterized the OPB domain and determined a minimal region for peptide design to suppress cancer cells. METHODS: Using alanine-scan method, we identified that the amino acids at OPB C-terminal are essential to YY1 binding to AKT. Further studies suggested that serine and threonine residues, but not lysines, in OPB play a key role in YY1-AKT interaction. We generated GFP fusion expression vectors to express OPB peptides with serially deleted N-terminal and found that OPB1 (i.e. G201-S226) is cytoplasmic, but OPB2 (i.e. E206-S226), OPB3 (i.e. E206-S226) and control peptide were both nuclear and cytoplasmic. RESULTS: Both OPB1 and 2 inhibited breast cancer cell proliferation and migration, but OPB3 exhibited similar effects to control. OPB1 and 2 caused cell cycle arrest at G1 phase, increased p53 and p21 expression, and reduced AKT(S473) phosphorylation in MCF-7 cells, but not in MDA-MB-231 cells. CONCLUSION: Overall, the serines and threonines of OPB are essential to YY1 binding to oncoproteins, and OPB peptide can be minimized to E206-S226 that maintain inhibitory activity to YY1- promoted cell proliferation.

A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily.

The short chain dehydrogenase/reductase superfamily (SDR) is a large family of NAD(P)H-dependent enzymes found in all kingdoms of life. SDRs are particularly well-represented in plants, playing diverse roles in both primary and secondary metabolism. In addition, some plant SDRs are also able to catalyse a reductive cyclisation reaction critical for the biosynthesis of the iridoid backbone that contains a fused 5 and 6-membered ring scaffold. Mining the EST database of Plantago major, a medicinal plant that makes iridoids, we identified a putative 5ß-progesterone reductase gene, PmMOR (P. major multisubstrate oxido-reductase), that is 60% identical to the iridoid synthase gene from Catharanthus roseus. The PmMOR protein was recombinantly expressed and its enzymatic activity assayed against three putative substrates, 8-oxogeranial, citral and progesterone. The enzyme demonstrated promiscuous enzymatic activity and was able to not only reduce progesterone and citral, but also to catalyse the reductive cyclisation of 8-oxogeranial. The crystal structures of PmMOR wild type and PmMOR mutants in complex with NADP+ or NAD+ and either 8-oxogeranial, citral or progesterone help to reveal the substrate specificity determinants and catalytic machinery of the protein. Site-directed mutagenesis studies were performed and provide a foundation for understanding the promiscuous activity of the enzyme.

Critical role of the pore domain in the cold response of TRPM8 channels identified by ortholog functional comparison.

In mammals, the main molecular entity involved in innocuous cold transduction is TRPM8. This polymodal ion channel is activated by cold, cooling compounds such as menthol and voltage. Despite its relevance, the molecular determinants involved in its activation by cold remain elusive. In this study we explored the use of TRPM8 orthologs with different cold responses as a strategy to identify new molecular determinants related with their thermosensitivity. We focused on mouse TRPM8 (mTRPM8) and chicken TRPM8 (cTRPM8), which present complementary thermosensitive and chemosensitive phenotypes. Although mTRPM8 displays larger responses to cold than cTRPM8 does, the avian ortholog shows a higher sensitivity to menthol compared with the mouse channel, in both HEK293 cells and primary somatosensory neurons. We took advantage of these differences to build multiple functional chimeras between these orthologs, to identify the regions that account for these discrepancies. Using a combination of calcium imaging and patch clamping, we identified a region encompassing positions 526-556 in the N terminus, whose replacement by the cTRPM8 homolog sequence potentiated its response to agonists. More importantly, we found that the characteristic cold response of these orthologs is due to nonconserved residues located within the pore loop, suggesting that TRPM8 has evolved by increasing the magnitude of its cold response through changes in this region. Our results reveal that these structural domains are critically involved in cold sensitivity and functional modulation of TRPM8, and support the idea that the pore domain is a key molecular determinant in temperature responses of this thermo-transient receptor potential (TRP) channel.

Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus.

Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.

A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity.

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid.

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis ), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis ) or linoleic acid (18:2Δ9cis,12cis ). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

An Iranian genomic sequence of Beet mosaic virus provides insights into diversity and evolution of the world population.

Beet mosaic virus (BtMV), the only Potyvirus known to infect sugar beet, occurs worldwide in beet crops. The full genome sequencing of a BtMV isolate from Iran (Ir-VRU), enabled us to better understand the evolutionary history of this virus. Selection analysis suggested that BtMV evolution is mainly under negative selection but its strength varies in different proteins with the multifunctional proteins under strongest selection. Recombination has played a major role in the evolution of the BtMVs; only the Ir-VRU and USA isolates show no evidence of recombination. The ML phylogenies of BtMVs from coat protein and full sequences were completely congruent. The primary divergence of the BtMV phylogeny is into USA and Eurasian lineages, and the latter then divides to form a cluster only found in Iran, and a sister cluster that includes all the European and Chinese isolates. A simple patristic dating method estimated that the primary divergence of the BtMV population was only 360 (range 260-490) years ago, suggesting an emergence during the development of sugar beet as a crop over the past three centuries rather than with the use of leaf beet as a vegetable for at least 2000 years.

Divergence of a Tandem Duplication of Manganese Superoxide Dismutase in Nosema bombycis.

Manganese superoxide dismutase (MnSOD) is a key enzyme in the protection of cells from oxidative stress. A tandem duplication of the MnSOD gene (NbMnSOD1 and NbMnSOD2) in the genome of Nosema bombycis, a parasite of the silkworm Bombyx mori, was previously identified. Here, we compare the protein structures of NbMnSOD1 and NbMnSOD2 and characterize these two proteins in terms of cellular localization, timing of transcription, protein structure, and enzyme activity. Despite a similarity in the primary sequence of NbMnSOD1 and NbMnSOD2, the latter shows a remarkable degree of amino acid sequence difference on the protein's surface and in the active site, where there is a substitution of a phenylalanine for a histidine in NbMnSOD2. Immuno-electron microscopy demonstrates that NbMnSOD1 is present in the cytosol of mature spores, whereas NbMnSOD2 is localized on the polar tube and the spore wall. Immunofluorescence confirms the localization of NbMnSOD2 on the polar tube of the germinated spore. Quantitative measurement of gene expression (qRT-PCR) demonstrates production of both alleles during the first day of infection followed by a dramatic decrease during the second to fourth day of infection. From the fifth day onward, the two alleles show a complementary pattern of expression. The qRT-PCR of the host manganese superoxide dismutase (BmMnSOD) shows a notable increase in transcription upon infection, leading to a three-fold spike by the first day of infection, followed by a decrease in transcription. Measurement of overall MnSOD activity shows a similar peak at day 1 followed by a decrease to a constant rate of enzyme activity. The differences in cellular localization and pattern of gene expression of NbMnSOD2 compared to NbMnSOD1, as well as the differences in protein structure seen for NbMnSOD2 compared to other microsporidial MnSODs, strongly suggest a unique, recently evolved role for NbMnSOD2.