Δ 9 -Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.natural products | phytocannabinoid | terpenophenolic | aldolase | ferredoxin-like
Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol are probably crucial in maintaining plant health during stress. Succinic semialdehyde (SSA) is a mitochondrially-generated intermediate in the metabolism of γ-aminobutyrate (GABA), which accumulates in response to a variety of biotic and abiotic stresses. SSA can be reduced to γ-hydroxybutyrate (GHB) under oxygen deficiency and high light conditions. Recent evidence indicates that distinct cytosolic and plastidial glyoxylate reductase isoforms from Arabidopsis (designated hereinafter as AtGR1 and AtGR2, respectively) catalyse the in vitro conversion of SSA to GHB, as well as glyoxylate to glycolate, via NADPH-dependent reactions. In the present report, the responses of GHB and related amino acids, as well as NADP+ and NADPH, were monitored in leaves from Arabidopsis or tobacco plants subjected to various abiotic stresses (i.e. Arabidopsis during exposure to salinity, drought, submergence, cold, or heat; tobacco during exposure to, and recovery from, submergence). Time-course experiments revealed that GHB accumulated in both Arabidopsis and tobacco plants subjected to stress, and that this accumulation was generally accompanied by higher GABA and alanine levels, higher NADPH/NADP+ ratio, and lower glutamate levels. Furthermore, the analysis of gene expression in Arabidopsis revealed that the relative abundance of GR1 (salinity, drought, submergence, cold, and heat) and GR2 (cold and heat) transcripts was enhanced by the stress tested. Thus, GHB accumulation in plants is a general response to abiotic stress and appears to be regulated by both biochemical and transcriptional processes.
γ-Aminobutyrate transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, the previously identified Arabidopsis thaliana (L.) Heyhn GABA-T (AtGABA-T) was characterized in more detail. Full-length AtGABA-T contains an N-terminal 36 amino acid long targeting pre-sequence (36 amino acids) that is both sufficient and necessary for targeting the enzyme to mitochondria. Removal of the pre-sequence encoding this N-terminal targeting domain and co-expression of the resulting truncated AtGABA-T cDNA with the GroES/EL molecular chaperone complex in Escherichia coli yielded good recovery of the soluble recombinant proteins. Activity assays indicated that purified recombinant GABA-T has both pyruvate- and glyoxylate-dependent activities, but cannot utilize 2-oxoglutarate as amino acceptor. Kinetic parameters for glyoxylate- and pyruvate-dependent GABA-T activities were similar, with physiologically relevant affinities. Assays of GABA-T activity in cell-free leaf extracts from wild-type Arabidopsis and two knockout mutants in different genetic backgrounds confirmed that the native enzyme possesses both pyruvate- and glyoxylate-dependent activities. The GABA-T transcript was present throughout the plant, but its expression was highest in roots and increased as a function of leaf development. A GABA-T with dual functions suggests the potential for interaction between GABA metabolism and photorespiratory glyoxylate production.
Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (Km glyoxylate=34 μM), and SSA to γ-hydroxybutyrate (Km SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (kcat/Km). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response.
Constitutive expression of an Arabidopsis thaliana (L.) Heynh cDNA (GenBank accession No. AY044183 ) in a succinic semialdehyde (SSA) dehydrogenase-deficient yeast ( Saccharomyces cerevisiae Hansen) mutant enables growth on γ-aminobutyrate and significantly enhances the accumulation of γ-hydroxybutyrate. In this report, the cDNA (designated hereinafter as AtGR1) was functionally expressed in Escherichia coli , and the recombinant protein purified to homogeneity. Kinetic analysis of substrate specificity revealed that the enzyme catalyzed the conversion of glyoxylate to glycolate (Km, glyoxylate = 4.5 μmol·L–1) as well as SSA to γ-hydroxybutyrate (Km, SSA = 0.87 mmol·L–1) via an essentially irreversible, NADPH-based mechanism. The enzyme had a 250-fold higher preference for glyoxylate than SSA based on the performance constants (kcat/Km), and with the exception of 4-carboxybenzaldehyde, at least a 100-fold higher preference for SSA than all other substrates tested (formaldehyde, acetaldehyde, butyraldehyde, 2-carboxybenzaldehyde, glyoxal, methylglyoxal, phenylglyoxal, phenylglyoxylate). In vitro assays of SSA reductase activity in cell-free extracts from Arabidopisis revealed its presence throughout the plant, although its specific activity was considerably higher in leaves at all developmental stages and in reproductive parts than in roots. It is proposed that the enzyme functions in redox homeostasis and the detoxification of both glyoxylate and SSA, in planta, resulting in the production of glycolate and γ-hydroxybutyrate, respectively.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.