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Direct conversion of carlactonoic acid to orobanchol by cytochrome P450 CYP722C in strigolactone biosynthesis.

ABSTRACT: Strigolactones (SLs) are carotenoid-derived phytohormones and rhizosphere signaling molecules for arbuscular mycorrhizal fungi and root parasitic weeds. Why and how plants produce diverse SLs are unknown. Here, cytochrome P450 CYP722C is identified as a key enzyme that catalyzes the reaction of BC-ring closure leading to orobanchol, the most prevalent canonical SL. The direct conversion of carlactonoic acid to orobanchol without passing through 4-deoxyorobanchol is catalyzed by the recombinant enzyme. By knocking out the gene in tomato plants, orobanchol was undetectable in the root exudates, whereas the architecture of the knockout and wild-type plants was comparable. These findings add to our understanding of the function of the diverse SLs in plants and suggest the potential of these compounds to generate crops with greater resistance to infection by noxious root parasitic weeds.

SUBMITTER: Wakabayashi T

PROVIDER: S-EPMC6989309 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Strigolactone biosynthesis catalyzed by cytochrome P450 and sulfotransferase in sorghum.

Project description:Root parasitic plants such as Striga, Orobanche, and Phelipanche spp. cause serious damage to crop production world-wide. Deletion of the Low Germination Stimulant 1 (LGS1) gene gives a Striga-resistance trait in sorghum (Sorghum bicolor). The LGS1 gene encodes a sulfotransferase-like protein, but its function has not been elucidated. Since the profile of strigolactones (SLs) that induce seed germination in root parasitic plants is altered in the lgs1 mutant, LGS1 is thought to be an SL biosynthetic enzyme. In order to clarify the enzymatic function of LGS1, we looked for candidate SL substrates that accumulate in the lgs1 mutants and performed in vivo and in vitro metabolism experiments. We found the SL precursor 18-hydroxycarlactonoic acid (18-OH-CLA) is a substrate for LGS1. CYP711A cytochrome P450 enzymes (SbMAX1 proteins) in sorghum produce 18-OH-CLA. When LGS1 and SbMAX1 coding sequences were co-expressed in Nicotiana benthamiana with the upstream SL biosynthesis genes from sorghum, the canonical SLs 5-deoxystrigol and 4-deoxyorobanchol were produced. This finding showed that LGS1 in sorghum uses a sulfo group to catalyze leaving of a hydroxyl group and cyclization of 18-OH-CLA. A similar SL biosynthetic pathway has not been found in other plant species.

| S-EPMC9292024 | biostudies-literature

Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis.

Project description:Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.

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Cytochrome P450 enzymes

Project description:Physiological plasticity in a polluted system: A case of an uncultured bacterium and its cypome

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Cytochrome P450 oxygenases of Taxol biosynthesis.

Project description:Cytochrome P450 monooxygenases play a prominent role in the biosynthesis of the diterpenoid anticancer drug Taxol, as they appear to constitute about half of the 19 enzymatic steps of the pathway in yew (Taxus) species. A combination of classical biochemical and molecular methods, including cell-free enzyme studies and differential-display of mRNA-reverse transcription polymerase chain reaction (RT-PCR) combined with a homology-based searching and random sequencing of a cDNA library from induced T. cuspidata cells, led to the discovery of six novel cytochrome P450 taxoid (taxane diterpenoid) hydroxylases. These genes show unusually high sequence similarity with each other (>70%) but low similarity (<30%) to, and significant evolutionary distance from, other plant P450s. Despite their high similarity, functional analysis of these hydroxylases demonstrated distinctive substrate specificities responsible for an early bifurcation in the biosynthetic pathway after the initial hydroxylation of the taxane core at C5, leading into a biosynthetic network of competing, but interconnected, branches. The use of surrogate substrates, in cases where the predicted taxoid precursors were not available, led to the discovery of two core oxygenases, the 2?- and the 7?-hydroxylase. This general approach could accelerate the functional analysis of candidate cDNAs from the extant family of P450 genes to identify the remaining oxygenation steps of this complex pathway.

| S-EPMC2901147 | biostudies-literature

Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation.

Project description:Cytochrome P450 monooxygenases (P450s) are considered nature's most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. KEY POINTS: •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

| S-EPMC8648635 | biostudies-literature

A cytochrome P450 CYP87A4 imparts sterol side-chain cleavage in digoxin biosynthesis

Project description:Identifying the foxglove P450scc through differential transcriptomic analysis.

2023-06-07 | GSE224014 | GEO

A key cytochrome P450 hydroxylase in pradimicin biosynthesis.

Project description:Pradimicins A-C (1-3) are a group of antifungal and antiviral polyketides from Actinomadura hibisca. The sugar moieties in pradimicins are required for their biological activities. Consequently, the 5-OH that is used for glycosylation plays a critical role in pradimicin biosynthesis. A cytochrome P450 monooxygenase gene, pdmJ, was amplified from the genomic DNA of A. hibisca and expressed in Escherichia coli BL21(DE3). PdmJ introduced a hydroxyl group to G-2A (4), a key pradimicin biosynthetic intermediate, at C-5 to form JX134 (5). A d-Ala-containing pradimicin analog, JX137a (6) was tested as an alternative substrate, but no product was detected by LC-MS, indicating that PdmJ has strict substrate specificity. Kinetic studies revealed a typical substrate inhibition of PdmJ activity. The optimal substrate concentration for the highest velocity is 115?M under the test conditions. Moreover, the conversion rate of 4 to 5 was reduced by the presence of 6, likely due to competitive inhibition. Coexpression of PdmJ and a glucose 1-dehydrogenase in E. coli BL21(DE3) provides an efficient method to produce the important intermediate 5 from 4.

| S-EPMC3248948 | biostudies-literature

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway.

Project description:We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA(12). A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA(12). Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase.

| S-EPMC29382 | biostudies-literature

Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis.

Project description:Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.

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The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid omega-hydroxylase involved in suberin monomer biosynthesis.

Project description:The lipophilic biopolyester suberin forms important boundaries to protect the plant from its surrounding environment or to separate different tissues within the plant. In roots, suberin can be found in the cell walls of the endodermis and the hypodermis or periderm. Apoplastic barriers composed of suberin accomplish the challenge to restrict water and nutrient loss and prevent the invasion of pathogens. Despite the physiological importance of suberin and the knowledge of the suberin composition of many plants, very little is known about its biosynthesis and the genes involved. Here, a detailed analysis of the Arabidopsis aliphatic suberin in roots at different developmental stages is presented. This study demonstrates some variability in suberin amount and composition along the root axis and indicates the importance of omega-hydroxylation for suberin biosynthesis. Using reverse genetics, the cytochrome P450 fatty acid omega-hydroxylase CYP86A1 (At5g58860) has been identified as a key enzyme for aliphatic root suberin biosynthesis in Arabidopsis. The corresponding horst mutants show a substantial reduction in omega-hydroxyacids with a chain length <C(20), demonstrating that CYP86A1 functions as a hydroxylase of root suberized tissue. Detailed expression studies revealed a strong root specificity and a localized expression in the root endodermis. Transgenic expression of CYP86A1 fused to GFP distributed CYP86A1 to the endoplasmic reticulum, indicating that suberin monomer biosynthesis takes place in this sub-cellular compartment before intermediates are exported in the apoplast.

| S-EPMC2423664 | biostudies-literature

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