Methylenedioxy Bridge Research Articles

Unraveling how hydrogen-bonding networks affect the capture of amphetamine-type stimulants by polymerized deep eutectic solvent modified magnetic biochar: Coupling quantum chemical calculations with experiment

The abuse of amphetamine-type stimulants (ATSs) has caused irreversible harm to public safety and ecosystems. A novel polymerized deep eutectic solvent modified magnetic pomelo peel biochar (PMBC) was prepared, and the differences in adsorption of four abused amphetamine-type stimulants (ATSs: AMP, MAMP, MDA and MDMA) were due to varying hydrogen bonds quantities and strengths. PMBC showed excellent chemical reactivity to MDMA, with a maximum adsorption capacity of 926.13 μg g−1, which was 3.25, 2.52 and 1.15 times higher than that of AMP, MAMP and MDA, respectively. Modern spectral analysis showed that there were a series of active centers (-COOH, -NH2 and -OH) on the PMBC, which could form hydrogen bond networks with the nitrogen and oxygen functional groups of ATSs. In various chemical environments: pH level (4–11), inorganic ion and organic matter (humic acid), PMBC maintained high activity towards four ATSs. Additionally, the quantum chemical calculations revealed that the methylenedioxy bridge of ATSs can increase the active sites, and the -NH- and -NH2 groups had different hydrogen bond formation capabilities, which together resulted in the adsorption order of PMBC on the four ATSs: MDMA > MDA > MAMP > AMP. Moreover, the hydrogen-bonding binding energies of several common hydrogen-bonding types were compared, including O-H····O, N-H····O/O-H····N and N-H···N. This study laid an empirical and theoretical foundation for the efficient capture of ATSs in water and contributed to the innovative design of materials.

Identification of the cytochrome P450s responsible for the biosynthesis of two types of aporphine alkaloids and their de novo biosynthesis in yeast.

Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category. In this study, we identified two specific cytochrome P450 enzymes (CYP80G6 and CYP80Q5) with distinct activities toward (S)-configured and (R)-configured substrates from the herbaceous perennial vine Stephania tetrandra, shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories. Additionally, we characterized two CYP719C enzymes (CYP719C3 and CYP719C4) that catalyzed the formation of the methylenedioxy bridge, an essential pharmacophoric group, on the A- and D-rings, respectively, of aporphine alkaloids. Leveraging the functional characterization of these crucial cytochrome P450 enzymes, we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast (Saccharomyces cerevisiae) for the de novo production of compounds such as (R)-glaziovine, (S)-glaziovine, and magnoflorine. This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.

A biosynthetic network for protoberberine production in Coptis chinensis.

Protoberberine alkaloids are a group of tetracyclic isoquinoline compounds known for their well-established antimicrobial and anti-inflammatory properties. The richness and diversity of protoberberine alkaloids accumulated in the Coptis genus necessitate a comprehensive examination of the biosynthetic machinery to understand their ecological significance. Here, from Coptis chinensis we identified CcCYP719A1, which could install a methylenedioxy bridge on either ring A or ring D of the protoberberine backbone, thus diverging metabolite flux towards the biosynthesis of various protoberberine components. We also obtained CcCYP719A2 and CcCYP719A3, which underwent positive selection after diverging from CcCYP719A1 and maintained specific catalytic activity on ring D. Further, we resolved the biosynthetic pathway of jatrorrhizine by identifying two demethylases, which could also modulate protoberberine composition by removing the C-3 methyl group and methylenedioxy bridge of ring D, allowing demethylated metabolites to be redirected into different routes. Moreover, we characterized 2-O-methyltransferase CcOMT1 and flavin-dependent oxidase CcTHBO, respectively responsible for the commonly observed 2-O-methylation and aromatic ring-C assembly in protoberberine alkaloids. Overall, this study reveals an interconnected metabolite network from which diverse protoberberine alkaloids originate. It provides valuable insights into the existence of undiscovered protoberberine components, and paves the way for the targeted production of desired protoberberine components for potential therapeutic development.

Elucidation of the (R)-enantiospecific benzylisoquinoline alkaloid biosynthetic pathways in sacred lotus (Nelumbo nucifera)

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant specialized metabolites found mainly in members of the order Ranunculales, including opium poppy (Papaver somniferum), for which BIA biosynthetic pathways leading to the critical drugs morphine, noscapine, and sanguinarine have been elucidated. Sacred lotus (Nelumbo nucifera), in the order Proteales, accumulates medicinal BIAs in the proaporphine, aporphine, and bisbenzylisoquinoline structural subgroups with a prevalence of R enantiomers, opposed to the dominant S configuration occurring in the Ranunculales. Nevertheless, distinctive BIA biosynthetic routes in sacred lotus have not been explored. In planta labeling experiments and in vitro assays with recombinant enzymes and plant protein extracts showed that dopamine and 4-hydroxyphenylacetaldehyde derived from l-tyrosine serve as precursors for the formation of (R,S)-norcoclaurine in sacred lotus, whereas only (R)-norcoclaurine byproducts are favored in the plant by action of R-enantiospecific methyltransferases and cytochrome P450 oxidoreductases (CYPs). Enzymes responsible for the R-enantiospecific formation of proaporphine (NnCYP80Q1) and bisbenzylisoquinoline (NnCYP80Q2) scaffolds, and a methylenedioxy bridge introduction on aporphine substrates (NnCYP719A22) were identified, whereas additional aspects of the biosynthetic pathways leading to the distinctive alkaloid profile are discussed. This work expands the availability of molecular tools that can be deployed in synthetic biology platforms for the production of high-value alkaloids.

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production

Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.

Alkaloids and Styryl lactones from Goniothalamus ridleyi King and Their α-Glucosidase Inhibitory Activity.

Gonioridleylactam (1), a new compound, is a unique dimeric aristolactam isolated from the EtOAc extract of the twigs of Goniothalamus ridleyi King. The structure of gonioridleylactam (1) consists of two different aristolactams linked together with two methylenedioxy bridges at C-3/C-3' and C-4/C-4', generating a ten-membered ring of [1,3,6,8]tetraoxecine. A new natural product, gonioridleyindole (3-hydroxymethyl-1-methyl-1H-benz[f]indole-4,9-dione, 2), together with eight known compounds (3-10) were also isolated from this plant. Their structures were extensively characterized by spectroscopic methods and comparisons were made with the literature. Compounds 1-4, 7, and 9 were evaluated for their α-glucosidase inhibitory activity. Of these, 3,5-demethoxypiperolide (7) displayed the highest α-glucosidase inhibitory activity, with an IC50 value of 1.25 µM.

Molecular Basis of the Unusual Seven-Membered Methylenedioxy Bridge Formation Catalyzed by Fe(II)/α-KG-Dependent Oxygenase CTB9

Methylenedioxy bridges (MDBs) are architecturally important motifs in natural products and bioactive molecules. Cercosporin, a typical perylenequinone pigment, contains an unusual seven-membered MDB, which has versatile biological and photocatalytic activities. Although cercosporin has been isolated, characterized, and studied for several decades, its biosynthetic pathway, especially the formation of the seven-membered MDB, has remained unclear. Here, we show that the formation of the seven-membered MDB is catalyzed by Fe(II)/α-ketoglutaric acid (α-KG)-dependent dioxygenase CTB9 in cercosporin biosynthesis. Moreover, crystal structures of CTB9 in complex with an α-KG analogue NOG (CTB9·Cu·NOG) and its substrate pre-cercosporin with NOG (CTB9·Cu·NOG·pre-cercosporin) were determined. These structures, together with site-directed mutagenesis studies and quantum mechanics calculations, help define the mechanism of the unique seven-membered MDB in cercosporin biosynthesis. In summary, these results provide molecular insights into other biosynthetic pathways of natural products containing MDBs.

Piper nigrum CYP719A37 Catalyzes the Decisive Methylenedioxy Bridge Formation in Piperine Biosynthesis.

Black pepper (Piper nigrum) is among the world’s most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.

Hancockiamides: phenylpropanoid piperazines from Aspergillus hancockii are biosynthesised by a versatile dual single-module NRPS pathway.

The hancockiamides are an unusual new family of N-cinnamoylated piperazines from the Australian soil fungus Aspergillus hancockii. Genomic analyses of A. hancockii identified a biosynthetic gene cluster (hkm) of 12 genes, including two single-module nonribosomal peptide synthetase (NRPS) genes. Heterologous expression of the hkm cluster in A. nidulans confirmed its role in the biosynthesis of the hancockiamides. We further demonstrated that a novel cytochrome P450, Hkm5, catalyses the methylenedioxy bridge formation, and that the PAL gene hkm12 is dispensable, but contributes to increased production of the cinnamoylated hancockiamides. In vitro enzymatic assays and substrate feeding studies demonstrated that NRPS Hkm11 activates and transfers trans-cinnamate to the piperazine scaffold and has flexibility to accept bioisosteric thienyl and furyl analogues. This is the first reported cinnamate-activating fungal NRPS. Expression of a truncated cluster lacking the acetyltransferase gene led to seven additional congeners, including an unexpected family of 2,5-dibenzylpiperazines. These pleiotropic effects highlight the plasticity of the pathway and the power of this approach for accessing novel natural product scaffolds.

Assembly of Plant Enzymes in E. coli for the Production of the Valuable (-)-Podophyllotoxin Precursor (-)-Pluviatolide.

Lignans are plant secondary metabolites with a wide range of reported health-promoting bioactivities. Traditional routes toward these natural products involve, among others, the extraction from plant sources and chemical synthesis. However, the availability of the sources and the complex chemical structures of lignans often limit the feasibility of these approaches. In this work, we introduce a newly assembled biosynthetic route in E. coli for the efficient conversion of the common higher-lignan precursor (+)-pinoresinol to the noncommercially available (-)-pluviatolide via three intermediates. (-)-Pluviatolide is considered a crossroad compound in lignan biosynthesis, because the methylenedioxy bridge in its structure, resulting from the oxidation of (-)-matairesinol, channels the biosynthetic pathway toward the microtubule depolymerizer (-)-podophyllotoxin. This oxidation reaction is catalyzed with high regio- and enantioselectivity by a cytochrome P450 monooxygenase from Sinopodophyllum hexandrum (CYP719A23), which was expressed and optimized regarding redox partners in E. coli. Pinoresinol-lariciresinol reductase from Forsythia intermedia (FiPLR), secoisolariciresinol dehydrogenase from Podophyllum pleianthum (PpSDH), and CYP719A23 were coexpressed together with a suitable NADPH-dependent reductase to ensure P450 activity, allowing for four sequential biotransformations without intermediate isolation. By using an E. coli strain coexpressing the enzymes originating from four plants, (+)-pinoresinol was efficiently converted, allowing the isolation of enantiopure (-)-pluviatolide at a concentration of 137 mg/L (ee ≥99% with 76% isolated yield).

Uncovering the cytochrome P450-catalyzed methylenedioxy bridge formation in streptovaricins biosynthesis

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.

Axially Chiral peri-Xanthenoxanthenes as a Circularly Polarized Luminophore.

A series of axially chiral peri-xanthenoxanthenes (PXXs) were synthesized from 1,1'-bi-2-naphthol without optical resolution. These are the first examples of chiral PXXs. Among them, PXX 5 with two naphthalene rings connected via a methylenedioxy bridge and a chiral axis displayed intense fluorescence (FL) and circularly polarized luminescence (CPL) both in solution (ΦFL of 0.72 and |glum| of 1.4 × 10-3) and in the solid state (ΦFL of 0.13 and |glum| of 4.8 × 10-3). The bridged structure of PXX 5 adjusts the angle between the vectors of the electric and magnetic transition dipole moments, which is essential for showing intense CPL activity.

Structure-activity studies on analogues of 4-methylguaiacol, a cattle anal odour constituent repellent to the brown ear tick (Rhipicephalus appendiculatus)

Previously, 4-methylguaiacol, a major constituent of cattle anal odour, was found to have a high repellency on Rhipicephalus appendiculatus. In the present study, 10 structural analogues of the phenol were tested for repellency against R. appendiculatus in order to assess the effects of (i) absence or presence of the 4-alkyl group of varying length, (ii) inclusion of a double bond in the 4-alkyl chain, (iii) linking the two phenolic oxygen in a methylenedioxy bridge, (iv) replacement of the OCH3 with CH3 and inclusion of another CH3 at position 6, and (v) presence of an additional OCH3 group at position 6. The analogues comprised of 2-methoxyphenol (guaiacol), 4-ethyl-2-methoxyphenol, 4-propyl-2-methoxyphenol, 4-allyl-2-methoxyphenol (eugenol), 3,4-methylenedioxytoluene, 2,4-dimethylphenol, 4-ethyl-2-methylphenol, 2,4,6-trimethylphenol, 4-propyl-2,6-dimethoxy-phenol and 4-allyl-2,6-dimethoxyphenol, which were compared at different concentrations in a two-choice climbing assay set up. Each analogue showed either increased or reduced repellency compared with 4- methylguaiacol. The structural feature that was associated with the highest repellency was 4-propyl moiety in the guaiacol unit (RD75 = 0.031 for 4-propyl-2-methoxyphenol; that of 4-methylguaiacol = 0.564). Effects of blending selected analogues with high repellency were also compared. However, none of the blends showed incremental increases in repellency compared with that of 4-propyl-2-methoxyphenol. We are currently evaluating the effects of controlled release of this compound at different sites on cattle on the behavior and success of R. appendiculatus to locate their predilection for feeding sites.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains.

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

Formation of a Methylenedioxy Bridge in (+)-Epipinoresinol by CYP81Q3 Corroborates with Diastereomeric Specialization in Sesame Lignans.

Plant specialized metabolites are often found as lineage-specific diastereomeric isomers. For example, Sesamum alatum accumulates the specialized metabolite (+)-2-episesalatin, a furofuran-type lignan with a characteristic diastereomeric configuration rarely found in other Sesamum spp. However, little is known regarding how diastereomeric specificity in lignan biosynthesis is implemented in planta. Here, we show that S. alatum CYP81Q3, a P450 orthologous to S. indicum CYP81Q1, specifically catalyzes methylenedioxy bridge (MDB) formation in (+)-epipinoresinol to produce (+)-pluviatilol. Both (+)-epipinoresinol and (+)-pluviatilol are putative intermediates of (+)-2-episesalatin based on their diastereomeric configurations. On the other hand, CYP81Q3 accepts neither (+)- nor (-)-pinoresinol as a substrate. This diastereomeric selectivity of CYP81Q3 is in clear contrast to that of CYP81Q1, which specifically converts (+)-pinoresinol to (+)-sesamin via (+)-piperitol by the sequential formation of two MDBs but does not accept (+)-epipinoresinol as a substrate. Moreover, (+)-pinoresinol does not interfere with the conversion of (+)-epipinoresinol to (+)-pluviatilol by CYP81Q3. Amino acid substitution and CO difference spectral analyses show that polymorphic residues between CYP81Q1 and CYP81Q3 proximal to their putative substrate pockets are crucial for the functional diversity and stability of these two enzymes. Our data provide clues to understanding how the lineage-specific functional differentiation of respective biosynthetic enzymes substantiates the stereoisomeric diversity of lignan structures.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Functional Analysis of Cytochrome P450s Involved in Streptovaricin Biosynthesis and Generation of Anti-MRSA Analogues.

The streptovaricins, chemically related to the rifamycins, are highly effective antibacterial agents, particularly against mycobacteria. Herein, a bioassay-guided investigation of Streptomyces spectabilis CCTCC M2017417 has led to the characterization of streptovaricins as potent compounds against methicillin-resistant Staphylococcus aureus (MRSA). We identified the streptovaricin biosynthetic gene cluster from S. spectabilis CCTCC M2017417 based on genomic sequencing and bioinformatic analysis. Targeted in-frame deletion of five cytochrome P450 genes (stvP1-P5) resulted in the identification of four new streptovaricin analogues and revealed the functions of these genes as follows: stvP1, stvP4, and stvP5 are responsible for the hydroxylation of C-20, Me-24, and C-28, respectively. stvP2 is possibly involved in formation of the methylenedioxy bridge, and stvP3, a conserved gene found in the biosynthetic cluster for naphthalenic ansamycins, might be related to the formation of a naphthalene ring. Biochemical verification of the hydroxylase activity of StvP1, StvP4, and StvP5 was performed, and StvP1 showed unexpected biocatalytic specificity and promiscuity. More importantly, anti-MRSA studies of streptovaricins and derivatives revealed significant structure-activity relationships (SARs): The hydroxyl group at C-28 plays a vital role in antibacterial activity. The hydroxyl group at C-20 substantially enhances activity in the absence of the methoxycarbonyl side chain at C-24, which can increase the activity regardless of the presence of a hydroxyl group at C-20. The inner lactone ring between C-21 and C-24 shows a positive effect on activity. This work provides meaningful information on the SARs of streptovaricins and demonstrates the utility of the engineering of streptovaricins to yield novel anti-MRSA molecules.

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.

Piperine and Derivatives: Trends in Structure-Activity Relationships.

Piperine is the main constituent of pepper, a commonly used kitchen spice and has been reported to possess various pharmacological activities. The structural features, an aromatic ring with a methylenedioxy bridge, a conjugated dienone system and a piperidine ring constituting an amide bond, possessed by the molecule have been considered important for the molecule to exhibit an array of bioactivities. Several modifications of above structural units have affected the biological properties of piperine, either enhancing or in some cases completely abolishing the activity. The present review emphasizes on the synthetic aspects of piperine along with the structure-activity relationships of its derivatives so as to rationalize the discovery of newer piperine based molecules.

Mode-of-action and evolution of methylenedioxy bridge forming P450s in plant specialized metabolism

(+)-Sesamin is a major furofran-class lignan in sesame seeds and harbors characteristic two methylenedioxy bridges (MDB) that are sequentially formed from (+)-pinoresinol via (+)-piperitol by a Sesamum indicum P450, CYP81Q1. However, the molecular basis for this unique catalytic activity of CYP81Q1 has been poorly understood. To elucidate MDB formation, we tested various natural and non-natural metabolites as substrates for CYP81Q1. A synthetic (+)-SC1mr and a naturally occurring (+)-kobusin showed inhibitory effect on the production of (+)-sesamin by CYP81Q1 unlike (+)-epipinoresinol and (−)-pinoresinol, indicating the strict diastereomer and enantiomer selectivity. Homology modeling followed by site-directed mutagenesis of CYP81Q1 showed that an amino acid residue crucial for MDB formation is a unique Ala residue (A308), located in I-helix proximal to the substrate pocket, responsible to the conserved distal-Thr residue. MDB by CYP81Q1 is produced possibly through the formation of a substrate-participated hydrogen-bonding network, since single replacement of the Ala by Thr severely and specifically lowered the MDB forming activity. This hypothesis is supported by a newly identified MDB-forming enzyme CYP81Q38 from Phryma leptostachya harboring an Ala responsible to Ala308 in CYP81Q1. An evolutional perspective of CYP81Q1 is discussed in relation to another MDB-forming CYP719As functionally conserved in Ranunculales.