Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta.

Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.

In silico identification and structure function analysis of a putative coclaurine N-methyltransferase from Aristolochia fimbriata.

In this study we isolated and performed in silico analysis of a putative coclaurine N-methyltransferase (CNMT) from the basal angiosperm Aristolochia fimbriata. The Aristolochiaceae plant family produces alkaloids similar to the Papavaraceae family, and CNMTs are central enzymes in biosynthesis pathways producing compounds of ethnopharmacological interest. We used bioinformatics and computational tools to predict a three-dimensional homology model and to investigate the putative function of the protein and its mechanism for methylation. The putative CNMT is a unique (S)-adenosyl-L-methionine (SAM)-dependent N-methyltransferase, catalyzing transfer of a methyl group from SAM to the amino group of coclaurine. The model revealed a mixed α/ß structure comprising seven twisted ß-strands surrounded by twelve α-helices. Sequence comparisons and the model indicate an N-terminal catalytic Core domain and a C-terminal domain, of which the latter forms a pocket for coclaurine. An additional binding pocket for SAM is connected to the coclaurine binding pocket by a small opening. CNMT activity is proposed to follow an SN2-type mechanism as observed for a similarly conformed enzyme. Residues predicted for the methyl transfer reaction are Tyr79 and Glu96, which are conserved in the sequence from A. fimbriata and in homologous N-methyltransferases. The isolated CNMT is the first to be investigated from any basal angiosperm.

An Enzyme from Aristolochia indica Destabilizes Fibrin-ß Amyloid Co-Aggregate: Implication in Cerebrovascular Diseases.

Fibrinogen and ß-amyloid (Aß) peptide independently form ordered aggregates but in combination, they form disordered structures which are resistant to fibrinolytic enzymes like plasmin and cause severity in cerebral amyloid angiopathy (CAA). A novel enzyme of 31.3 kDa has been isolated from the root of the medicinal plant Aristolochia indica that showed fibrinolytic as well as fibrin-Aß co-aggregate destabilizing properties. This enzyme is functionally distinct from plasmin. Thrombolytic action of the enzyme was demonstrated in rat model. The potency of the plant enzyme in degrading fibrin and fibrin-plasma protein (Aß, human serum albumin, lysozyme, transthyretin and fibronectin) co-aggregates was demonstrated by atomic force microscopy, scanning electron microscopy and confocal microscopy that showed better potency of the plant enzyme as compared to plasmin. Moreover, the plant enzyme inhibited localization of the co-aggregate inside SH-SY5Y human neuroblastoma cells and also co-aggregate induced cytotoxicity. Plasmin was inefficient in this respect. In the background of limited options for fragmentation of these co-aggregates, the plant enzyme may appear as a potential proteolytic enzyme.

Characterization of the aqueous extract of the root of Aristolochia indica: evaluation of its traditional use as an antidote for snake bites.

ETHNOPHARMACOLOGICAL RELEVANCE: The aqueous extract of the roots of Aristolochia indica is used as a decoction for the ailment of a number of diseases including snake bite treatment. Though the alcoholic extract of the different parts of the plant are well studied, information on the aqueous extract is limited. We have estimated aristolochic acid, different enzymes, enzyme inhibitors and anti-snake venom potency of its root extract. MATERIALS AND METHODS: Reverse phase-HPLC was used to quantify aristolochic acid. Zymography, DQ-gelatin assay and atomic force microscopy were done to demonstrate gelatinase and collagenase activities of the extract. SDS-PAGE followed by MS/MS analysis revealed the identity of major protein components. Toxicity of the extract was estimated on animal model. Interaction of the extract with Russell's viper venom components was followed by Rayleigh scattering and enzyme assay. RESULTS: The aristolochic acid content of the root extract is 3.08 ± 1.88 × 10(-3)mg/ml. The extract possesses strong gelatinolytic, collagenase, peroxidase and nuclease activities together with l-amino acid oxidase and protease inhibitory potencies. Partial proteomic studies indicated presence of starch branching enzymes as major protein constituent of the extract. The extract did not show any acute and sub-chronic toxicity in animals at lower doses, but high dose causes liver and kidney damage. The extract elongated duration of survival of animals after application of Russell's viper venom. CONCLUSIONS: Considering the low aristolochic acid content of the extract, its consumption for a short time at moderate dose does not appear to cause serious toxicity. Strong inhibition of l-amino acid oxidase may give partial relief from snake bite after topical application of the extract.

[Allozyme variation of the relict plant Aristolochia manshuriensis Kom. (Aristolochiaceae)].

Allozyme variation of a rare relict plant, birthwort Aristolochia manshuriensis Kom., was examined. The main parameters of genetic variation in natural populations of A. manshuriensis from the Anan'evka, Nezhinka, and Malaya Borisovka river basins (Primorskii Krai) were inferred from analysis of nine enzyme systems, presumably encoded by 18 loci. At 99% polymorphism criterion, 24.4% of A. manshuriensis loci were shown to be polymorphic. The mean number of alleles per locus was 1.24; the mean observed and expected heterozygosities, 0.12 and 0.10, respectively.