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PubMed1993

[Possibility of using flour of pigeon pea in products prepared with rice or wheat flour].

The present study reports on the development of foods containing processed pigeon pea (Cajanus cajan) flour. The pigeon pea flours described in a previous publication were prepared from dehulled pigeon peas by cooking in autoclave, by extrusion-cooking and by cooking/dehydration by drum-drying. Mixtures of cooked pigeon peas and rice were first evaluated biological through a protein complementation design using NPR. The results of this study showed that the two products had high protein quality and were similar when mixed in ratios of 80:20 to 40:60. For the evaluation of the processed pigeon pea flour, mixtures with rice (80:20) were used. All pigeon pea flours gave similar protein quality values. On the basis of these results three products were developed and tested. One was a gruel ("atole"), a second a fruit-flavored thick drink with and without 15% milk. Cookies were also prepared with a series of blends of pigeon pea flour (extrusion-cooked) and wheat. The gruel and the fruit flavored products had high acceptability based on a sensory evaluation test. Cookies with 100% pigeon pea flour were unacceptable, however, mixtures of 75% wheat flour and 25% pigeon pea flour gave cookies of attractive appearance and good taste. The study showed the possibility of preparing and utilizing tropical grain legume flours for food products of relatively high acceptability and nutritive value.

Fabaceae↗

PubMed2001

Pea varieties resources in Russia in context to the situation in Western Europe.

The largest share of legumes grain production in Russia falls at peas and amounts to 75%. The pea is grown practically in all agricultural regions of Russia. The most favourable pea-growing regions are the forest-steppe zone of European part of Russia and Volga region. 84 varieties of pea are being included in the official "National List of the Breeding Achievements Permitted to Use in the Russian Federation" in 2001. 24 of these varieties correspond to the requirements for the most valuable food pea ones. The pea varieties especially suitable for food are identified by a special classification. 32.5% percent of a total area sown with the peas in Europe falls to Russia. Total peas production in Russia is 10.6% of a total European production.

Cooking↗

PubMed1988

Identification of nodX, a gene that allows Rhizobium leguminosarum biovar viciae strain TOM to nodulate Afghanistan peas.

Gene(s) conferring the ability of Rhizobium leguminosarum biovar viciae strain TOM to nodulate primitive peas (cultivar Afghanistan) had been located in a 2.0 kb region of its sym plasmid, pRL5JI. In this DNA, a single open reading frame of 1101 bp, corresponding to a gene, nodX was found. nodX is downstream of nodJ which is present in strain TOM and also in the sym plasmid of a typical strain of this biovar. nodX specifies a hydrophobic protein (of Mr 41,036) with no clear similarity to other proteins in data bases. Mutations in nodX abolished nodulation of Afghanistan peas but not nodulation of commercial peas. nodX-lacZ fusions were used to show that transcription of nodX was activated by root exudates from both commercial and Afghanistan peas and by defined flavonoids. Exudate from Afghanistan peas activated nod genes of typical strains of R. leguminosarum biovar viciae which fail to nodulate these peas; thus, their failure to nodulate these primitive peas is not due to a lack of activation of their nod genes by exudate from Afghanistan peas. A homologue of nodX exists in R. leguminosarum biovar trifolii (which nodulates clover) but not in typical strains of R. leguminosarum biovar viciae.

Base Sequence↗

PubMed2004

Biochemical and immunological characterization of pea nuclear intermediate filament proteins.

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6-12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.

Amino Acid Sequence↗

PubMed1980

Transfer ribonucleic acid genes in the chloroplast deoxyribonucleic acid of pea leaves.

The saturation hybridization between pea ctDNA and 125I-labeled pea ct-tRNAs has shown that 1.2% of the peak ctDNA codes for tRNA genes. The observed level of hybridization has been found to result from specific base pairings between ctDNA and ct-RNA as shown by competition hybridization experiments and thermal stability studies on DNA-tRNA hybrids. The level of hybridization obtained in this study amounts to the presence of approximately 40 tRNA genes in pea ctDNA. The tRNAs from the cytoplasm of the pea leaves, Escherichia coli, yeast, and calf thymus did not compete with the pea ct-tRNAs for the common base sequences in pea ctDNA. The presence of 17 aminoacyl-tRNA synthetases and their corresponding tRNAs was demonstrated in chloroplast. The acylation of ct-tRNAs proceeds with the same rate whether the partially purified tRNA synthetases from chloroplasts of E. coli are used. The aminoacylation of the three amino acids glutamic acid, glutamine, and cysteine proceeded very slowly in chloroplasts. The individually labeled aminoacyl-tRNAs hybridized with pea ctDNA. The hybridization follows true saturation rates, and the melting profiles of aminoacyl-tRNA-ctDNA indicate the formation of specific base pairs between the ctDNA and tRNA. Seventeen aminoacyl-tRNA genes have been identified in the pea ctDNA.

Chloroplasts↗

PubMed2002

Microsynteny between pea and Medicago truncatula in the SYM2 region.

The crop legume pea (Pisum sativum) is genetically well characterized. However, due to its large genome it is not amenable to efficient positional cloning strategies. The purpose of this study was to determine if the model legume Medicago truncatula, which is a close relative of pea, could be used as a reference genome to facilitate the cloning of genes identified based on phenotypic and genetic criteria in pea. To this end, we studied the level of microsynteny between the SYM2 region of pea and the orthologous region in M. truncatula. Initially, a marker tightly linked to SYM2 was isolated by performing differential RNA display on near-isogenic pea lines. This marker served as the starting point for construction of a BAC physical map in M. truncatula. A fine-structure genetic map, based on eight markers from the M. truncatula physical map, indicates that the two genomes in this region share a conserved gene content. Importantly, this fine structure genetic map clearly delimits the SYM2-containing region in pea and the SYM2-orthologous region in M. truncatula, and should provide the basis for cloning SYM2. The utility of the physical and genetic tools in M. truncatula to dissect the SYM2 region of pea should have important implications for other gene cloning experiments in pea, in particular where the two genomes are highly syntenic within the region of interest.

Chromosome Mapping↗

PubMed2003

Pea lectin expressed transgenically in oilseed rape reduces growth rate of pollen beetle larvae.

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T0-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T2-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T2-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.

Animals↗

PubMed2005

Phosphorylation of phosphoprotein enriched in astrocytes (PEA-15) regulates extracellular signal-regulated kinase-dependent transcription and cell proliferation.

Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser104 and Ser116 is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15. Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP kinase signaling.

Active Transport, Cell Nucleus↗

PubMed1997

[15N]-labeled pea flour protein nitrogen exhibits good ileal digestibility and postprandial retention in humans.

The aim of the present study was to evaluate postprandial absorption of pea protein as well as exogenous nitrogen retention in humans. For this purpose, after fasting overnight, seven healthy adults (4 males and 3 females) ingested [15N]-labeled pea protein (195 mmol N). Ileal effluents were collected for 8 h at 30-min intervals using a nasointestinal intubation technique. Urine and plasma samples were collected for 24 h. The [15N]-enrichment was determined in the intestinal samples, in the plasma amino acids and urea as well as in the urinary urea and ammonia fractions. The true gastroileal absorption of pea protein was 89.4 +/- 1.1%. This absorption was correlated with a significant increase (P < 0.05) in [15N]-enrichment in the plasma amino acids and in the nitrogen incorporated into the body urea pool for 1 h following pea ingestion. The enrichment remained significantly higher than the basal values in these pools 24 h after pea ingestion. The recovery of total urinary exogenous nitrogen after 22 h was 31.1 +/- 9.3 mmol N. Moreover, the kinetics of [15N]-labeled pea amino acids deamination reached a plateau of 39 mmol. Under these conditions, pea nitrogen retention represented 78% of the absorbed dietary nitrogen in healthy humans. The present results demonstrate the good true nitrogen digestibility and retention of pea protein in humans.

Adult↗

PubMed1990

Development and Partial Characterization of Nearly Isogenic Pea Lines (Pisum sativum L.) that Alter Uptake Hydrogenase Activity in Symbiotic Rhizobium.

Some Rhizobium bacteria have H(2)-uptake (Hup) systems that oxidize H(2) evolved from nitrogenase in leguminous root nodules. Pea (Pisum sativum L.) cultivars ;JI1205' and ;Alaska' produce high Hup (Hup(++)) and moderate Hup (Hup(+)) phenotypes, respectively, in Rhizobium leguminosarum 128C53. The physiological significance and biochemical basis of this host plant genetic effect are unknown. The purpose of this investigation was to advance basic Hup studies by developing nearly isogenic lines of peas that alter Hup phenotypes in R. leguminosarum strains containing hup genes. Eight pairs of nearly isogenic pea lines that produce Hup(++) and Hup(+) phenotypes in R. leguminosarum 128C53 were identified in 173 F(2)-derived F(6) families produced from crosses between JI1205 and Alaska. Tests with the pea isolines and three strains of hup-containing R. leguminosarum showed that the isolines altered Hup activity significantly (P </= 0.05) in 19 of 24 symbiotic combinations. Analyses of Hup phenotypes in F(6) families, the F(1) population, and two backcrosses suggested involvement of a single genetic locus. Three of the eight pairs of isolines were identified as being suitable for physiological studies, because the two lines in each pair showed similar growth, N assimilation, and flowering traits under nonsymbiotic conditions. Tests of those lines under N(2)-dependent conditions with isogenic Hup(+) and negligible Hup (Hup(-)) mutants of R. leguminosarum 128C53 showed that, in symbioses with Hup(+) rhizobia, two out of three Hup(++) pea lines decreased N(2) fixation relative to Hup(+) peas. In one of those cases, however, the Hup(++) plant line also decreased fixation by Hup(-) rhizobia. When results were averaged across all rhizobia tested, Hup(+) pea isolines had 8.2% higher dry weight (P </= 0.05) and fixed 12.6% more N(2) (P </= 0.05) than Hup(++) isolines. Pea lines described here may help identify host plant factors that influence rhizobial Hup activity and should assist in clarifying how Hup systems influence other physiological processes.

Journal Article↗

PubMed2002

Distribution of the pea pathogenicity ( PEP) genes in the fungus Nectria haematococca mating population VI.

Previous studies identified a cluster of six genes that are expressed in the fungus Nectria haematococca mating population VI during infection of pea. Four of these genes were shown to contribute to pathogenicity on pea and were called PEP genes for pea pathogenicity. The cluster is located on a "conditionally dispensable" (CD) chromosome and has features similar to bacterial pathogenicity islands. In this study, the occurrence and location of members of the PEP cluster were analyzed in laboratory strains and nine pea pathogenic and 16 non-pea pathogenic field isolates of N. haematococca. Our results indicate that all pea-pathogenic isolates have homologues for all six genes present in the PEP cluster and the homologues appear to be clustered. PEP homologues are also present in isolates that are not pathogenic on pea, although none of these isolates have homologues of all six genes. In addition, PEP homologues are found in CD chromosomes and in other chromosomes. Isolates without PEP homologues are virulent on ripe tomato fruits and carrot roots, indicating that PEP genes are not required for pathogenicity on these hosts.

Ascomycota↗

PubMed2004

Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products.

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and analysis of a pea cDNA that is expressed in darkness and very rapidly induced by gibberellic acid.

Using cDNA representational difference analysis (cDNA RDA), we isolated a cDNA named GDA-1 from a cDNA library constructed with mRNA from short-day (SD) grown G2 pea apical tissue. The amino acid sequence deduced from GDA-1 shares partial identity with the B2 protein which is expressed during embryogenesis of carrot cells. Northern analysis showed that GDA-1 mRNA is abundant in SD-grown G2 pea apical buds. In long-day (LD) conditions, there was almost no detectable GDA-1 mRNA. When LD-grown G2 peas were kept in continuous darkness for 24 h, the GDA-1 mRNA content reached a level equivalent to about 50% of that in the SD samples. On the other hand, when SD-grown peas were transferred into the light for 24 h, the amount of hybridizable GDA-1 mRNA dropped to the same as that of LD-grown plants. GDA-1 expression was found to be independent of flower initiation time. GA3 application in vitro resulted in rapid accumulation of GDA-1 mRNA in LD-grown G2 pea apical buds, which is compatible with its delaying effect on apical senescence. Time-course experiments revealed that GDA-1 is induced within 15 min of GA3 application. Exogenous GA3 did not influence the expression of GDA-1 in SD-grown G2 peas. Since both photoperiod and GA induce the expression of GDA-1, we speculate that they may activate similar signal transduction pathways in G2 peas. Our work also shows that photoperiod may change the efficiency of gibberellin perception by plants.

Amino Acid Sequence↗

PubMed1985

Coupling factor activity of the purified pea mitochondrial F1-ATPase.

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.

Adenosine Triphosphate↗

PubMed2002

Two light-responsive elements of pea chloroplastic fructose-1,6-bisphosphatase gene involved in the red-light-specific gene expression in transgenic tobaccos.

The pea chloroplast fructose-1,6-bisphosphatase (FBPase) gene was cloned from a pea genomic library and sequenced. The gene contained three introns and four exons. Both in vitro and in vivo analyses of the promoter region of the gene were carried out simultaneously to elucidate the mechanisms of light-mediated gene expression. Two light-responsive elements were identified in gel mobility shift assays: a GT-1-like sequence for the binding of a GT-1-like factor (termed pea factor 1; PF1) and a binding site for a dark-specific factor (termed pea factor 2; PF2). The binding affinity of PF1 was higher in light-grown peas than in dark-grown peas and was affected by phosphorylation. The binding site was located at nucleotides (nt) -326 to -341. PF2 binding was dark-specific and the binding region was located upstream of the PF1-binding site (nt -492 to -412). In vivo experiments with transgenic tobacco plants suggested that the region between nt -411 and -272 contained a PF1-binding site that promoted light-mediated expression of the pea chloroplast FBPase. In contrast, the 81-bp region between nt -492 and -412, which is located further upstream than the PF1-binding site, negatively regulated light-mediated expression of FBPase. Moreover, activation of gene expression by the region (nt -411 to -272) contained a PF1-binding site that was sensitive to red-light irradiation, suggesting that the expression of the chloroplast FBPase was regulated by the phytochrome system. Interestingly, the binding region for the dark-specific factor (PF2; nt -492 to -412) not only repressed gene expression in the dark, but also acted as a light-dependent activating element of the chloroplast FBPase gene.

Base Sequence↗

PubMed1997

Characterization of a recombinant pea 5-aminolevulinic acid dehydratase and comparative inhibition studies with the Escherichia coli dehydratase.

Pea 5-aminolevulinic acid dehydratase (ALAD) was purified 200-fold from a recombinant overproducing strain of Escherichia coli, yielding an octameric enzyme with a specific activity of 280 units mg-1. Divalent metal ions were essential, Mg2+, Mn2+, and Co2+ ions all supporting activity, whereas Zn2+ ions could not. Equilibrium dialysis and atomic absorption studies revealed two Mg2+ ion binding sites per subunit. Pea ALAD bound the substrate 5-aminolevulinic acid covalently through a Schiff base at the P-site, electrospray mass spectrometry of the reduced enzyme-ALA Schiff base complex showing the presence of one P-site per subunit. The amino acid residue modified by ALA was identified by MALDI-MS and Edman sequencing as Lys-293, analogous to the active site Lys-247 of E. coli ALAD and Lys-252 of mammalian ALAD. Comparative studies of pea ALAD with E. coli ALAD using the inhibitors 3-acetyl-4-oxoheptane-1,7-dioic acid (AOHD) and succinylacetone (SA) indicated similar modes of inhibition, with the formation of a Schiff base complex between the inhibitors and the active site lysine. Studies with the ALA homolog, 4-amino-3-oxobutanoic acid (AOB), revealed that it is specific for the A-site of both the pea and E. coli ALADs. An interesting difference exists between the enzymes, however, pea ALAD being far more susceptible to inhibition with AOB than the E. coli enzyme. AOB bound 10 times better to the A-site of pea ALAD compared to the substrate, ALA. Despite the 2000 times lower Ki of AOB for pea ALAD, no abortive Schiff base intermediate, between enzyme-bound ALA at the P-site and AOB bound at the A-site, could be demonstrated.

Amino Acid Sequence↗

PubMed1998

Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics.

Two hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the Avall site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli-expressed cell lysates as a high yielding 12 kDa protein. Western blot analysis showed a positive reaction with antibodies against pea Trxs m and f and, like the parenteral pea Trx m, displayed an acidic pI (5.0) and a high thermal stability. In contrast, the opposite hybrid Trx f/m appeared in E. coli lysates as inclusion bodies, where it was detected by Western blot against pea Trx f antibodies as a 40 kDa protein. Trx f/m was very unstable, sensitive to heat denaturation, and could not be purified. Trx m/f showed a higher affinity for pea chloroplast fructose-1,6-bisphosphatase (FBPase) and a smaller Trx/FBPase saturation ratio than both parenterals; however, the FBPase catalytic rate was lower than that with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP-malate dehydrogenase. Computer-assisted models of pea Trxs m and f, and of the chimeric Trx m/f, showed a change in the orientation of the alpha 4-helix in the hybrid, which could explain the kinetic modifications with respect to Trxs m and f. We conclude that the stability of Trxs lies on the N-side of the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pl of the whole molecule. In contrast, the ability of FBPase binding and enzyme catalysis depends on the structure on the C-side of the regulatory cysteines.

Amino Acid Sequence↗

PubMed2001

Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase.

We have produced a model to define the linoleate-binding pocket of pea 9/13-lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron-coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13-lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null-effect of mutating pea 9/13-lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9-lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13-lipoxygenase and cucumber 13/9-lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9-lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate-binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate-binding to mammalian 5-, 12- and 15-lipoxygenases, would appear to be true also for linoleate-binding to plant 9- and 13-lipoxygenases.

Amino Acid Sequence↗