Two CYP71AJ enzymes function as psoralen synthase and angelicin synthase in the biosynthesis of furanocoumarins in Peucedanum praeruptorum Dunn.

KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.

Convergent evolution leading to the appearance of furanocoumarins in citrus plants.

Furanocoumarins are defense molecules mainly described in four plant families that are phylogenetically distant. Molecular characterization of the biosynthetic pathway has been started for many years in Apiaceae and Rutaceae. The results obtained thus far in Apiaceae indicated a major role of cytochromes P450 (P450s) in the CYP71 family. In the present work, we describe the importance of another subfamily of P450s, CYP82D, identified by using a deep analysis of the citrus (Rutaceae) genome and microarray database. CYP82D64 is able to hydroxylate xanthotoxin to generate 5-OH-xanthotoxin. Minor and limited amino acid changes in the CYP82D64 coding sequence between Citrus paradisi and Citrus hystrix provide the enzyme in the latter with the ability to hydroxylate herniarin, but with low efficiency. The kinetic constants of the enzyme are consistent with those of other enzymes of this type in plants and indicate that it may be the physiological substrate. The activity of the enzyme is identical to that of CYP71AZ6 identified in parsnip, showing possible evolutionary convergence between these two families of plants. It is highly possible that these molecules are derived from the synthesis of ubiquitous coumarins throughout the plant kingdom.

Cho Endonuclease Functions during DNA Interstrand Cross-Link Repair in Escherichia coli.

DNA interstrand cross-links are complex lesions that covalently link both strands of the duplex DNA. Lesion removal is proposed to be initiated via the UvrABC nucleotide excision repair complex; however, less is known about the subsequent steps of this complex repair pathway. In this study, we characterized the contribution of nucleotide excision repair mutants to survival in the presence of psoralen-induced damage. Unexpectedly, we observed that the nucleotide excision repair mutants exhibit differential sensitivity to psoralen-induced damage, with uvrC mutants being less sensitive than either uvrA or uvrB We show that Cho, an alternative endonuclease, acts with UvrAB and is responsible for the reduced hypersensitivity of uvrC mutants. We find that Cho's contribution to survival correlates with the presence of DNA interstrand cross-links, rather than monoadducts, and operates at a step after, or independently from, the initial incision during the global repair of psoralen DNA adducts from the genome. IMPORTANCE: DNA interstrand cross-links are complex lesions that covalently bind to both strands of the duplex DNA and whose mechanism of repair remains poorly understood. In this study, we show that Cho, an alternative endonuclease, acts with UvrAB and participates in the repair of DNA interstrand cross-links formed in the presence of photoactivated psoralens. Cho's contribution to survival correlates with the presence of DNA interstrand cross-links and operates at a step after, or independently from, the initial incision during the repair process.

Induction of direct repeat recombination by psoralen-DNA adducts in Saccharomyces cerevisiae: defects in DNA repair increase gene copy number variation.

Psoralen photoreaction produces covalent monoadducts and interstrand crosslinks in DNA. The interstrand DNA crosslinks are complex double strand lesions that require the involvement of multiple pathways for repair. Homologous recombination, which can carry out error-free repair, is a major pathway for crosslink repair; however, some recombination pathways can also produce DNA rearrangements. Psoralen photoreaction-induced recombination in yeast was measured using direct repeat substrates that can detect gene conversions, a form of conservative recombination, as well as deletions and triplications, which generate gene copy number changes. In repair-proficient cells the major products of recombination were gene conversions, along with substantial fractions of deletions. Deficiencies in DNA repair pathways increased non-conservative recombination products. Homologous recombination-deficient rad51, rad54, and rad57 strains had low levels of crosslink-induced recombination, and most products were deletions produced by single strand annealing. Nucleotide excision repair-deficient rad1 and rad2 yeast had increased levels of triplications, and rad1 cells had lower crosslink-induced recombination. Deficiencies in post-replication repair increased crosslink-induced recombination and gene copy number changes. Loss of REV3 function, in the error-prone branch, and of RAD5 and UBC13, in the error-free branch, produced moderate increases in deletions and triplications; rad18 cells, deficient in both post-replication repair sub-pathways, exhibited hyperrecombination, with primarily non-conservative products. Proper functioning of all the DNA repair pathways tested was required to maintain genomic stability and avoid gene copy number variation in response to interstrand crosslinks.

CYP98A22, a phenolic ester 3'-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens.

BACKGROUND: Furanocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the furanocoumarin biosynthetic pathway and might indirectly impact their synthesis. RESULTS: In this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and furanocoumarin. A detailed analysis indicates that both genes enhance the production of furanocoumarins but that CYP98A22, unlike CYP98A3, doesn't affect the synthesis of scopoletin. CONCLUSIONS: The overexpression of CYP98A22 positively impacts the concentration of furanocoumarins in R. graveolens. This gene is therefore a valuable tool to engineer plants with improved therapeutical values that might also be more resistant to phytophageous insects.

Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis.

The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14 min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete "metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.

Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells.

A significant level of correction of the mutation responsible for sickle cell anemia has been achieved in monkey COS-7 cells on a plasmid containing a beta-globin gene fragment. The plasmid was treated in vitro with a nucleic acid 'third strand' bearing a terminal photoreactive psoralen moiety that binds immediately adjacent to the mutant base pair. Following covalent attachment of the psoralen by monoadduct or diadduct formation to the mutant T-residue on the coding strand, the treated plasmid was transfected into the cells, which were then incubated for 48 h to allow the cellular DNA repair mechanisms to remove the photoadducts. Upon re-isolation and amplification of the transfected plasmid, sickle cell mutation correction, as determined by sequence analysis of both complementary strands, was established in a full 1%. This result encourages extension of the approach to correct the mutation directly on the chromosome.