Biosynthesis of an Anti-Addiction Agent from the Iboga Plant.

(-)-Ibogaine and (-)-voacangine are plant derived psychoactives that show promise as treatments for opioid addiction. However, these compounds are produced by hard to source plants, making these chemicals difficult for broad-scale use. Here we report the complete biosynthesis of (-)-voacangine, and de-esterified voacangine, which is converted to (-)-ibogaine by heating, enabling biocatalytic production of these compounds. Notably, (-)-ibogaine and (-)-voacangine are of the opposite enantiomeric configuration compared to the other major alkaloids found in this natural product class. Therefore, this discovery provides insight into enantioselective enzymatic formal Diels-Alder reactions.

Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the anti-addictive alkaloid ibogaine from Tabernanthe iboga.

Monoterpenoid indole alkaloids are a large (∼3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales. Tabernanthe iboga or iboga (Apocynaceae) is native to western equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with bringing iboga to the attention of Western medicine through his accidental discovery that iboga can alleviate opioid withdrawal symptoms. Since this observation, iboga has been investigated for its use in the general management of addiction. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these studies may help improve the ibogaine supply chain through synthetic biology approaches. Here, we used next-generation sequencing to generate the first iboga transcriptome and leveraged homology-guided gene discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase (N10OMT). Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) and incubation with putative precursors, along with HPLC-MS analysis, confirmed the predicted activities of both enzymes. Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues. These discoveries coupled with our publicly available iboga transcriptome will contribute to additional gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and to the development of ibogaine as a treatment for addiction.

Hemostatic potential of latex proteases from Tabernaemontana divaricata (L.) R. Br. ex. Roem. and Schult. and Artocarpus altilis (Parkinson ex. F.A. Zorn) Forsberg.

Pharmacological properties exhibited by latex of plants are due to various biologically active compounds including several proteolytic enzymes. Present study evaluates hemostatic potential of Tabernaemontana divaricata and Artocarpus altilis from Apocynaceae and Moraceae families respectively. The latex of these plants were initially subjected to dialysis and crude extracts were estimated for proteolytic activity using casein as the substrate. Mean caseinolytic activity for 100 µg of latex protein was found to be 56.16 ± 0.57 and 45 ± 0.3 U/h for T. divaricata and A. altilis respectively. Caseinolytic activity by both the plant extracts was higher than standard proteases, papain and trypsin. However the difference was significant (p < 0.05) with papain alone. Crude enzymes (CE) from both plants exhibited coagulant activity on human platelet poor plasma by recalcification time. A significant reduction in clotting time was exhibited by T. divaricata compared to A. altilis (p < 0.05). These results were further substantiated with fibrinogen agarose plate assay. Crude enzyme of both plants also hydrolyzed blood clot. Mean % of thrombolysis by T. divaricata was 80.75 ± 1.2 and that of A. altilis was 70.24 ± 1.52. Inhibition studies confirmed cysteine protease nature of CE. Comparative analysis revealed T. divaricata to be the best among the two for its hemostatic potential. This study scientifically validates the use of latex from these plants in the management of fresh cuts or wounds.

Structural basis of the unusual stability and substrate specificity of ervatamin C, a plant cysteine protease from Ervatamia coronaria.

Ervatamin C is an unusually stable cysteine protease from the medicinal plant Ervatamia coronaria belonging to the papain family. Though it cleaves denatured natural proteins with high specific activity, its activity toward some small synthetic substrates is found to be insignificant. The three-dimensional structure and amino acid sequence of the protein have been determined from X-ray diffraction data at 1.9 A (R = 17.7% and R(free) = 19.0%). The overall structure of ervatamin C is similar to those of other homologous cysteine proteases of the family, folding into two distinct left and right domains separated by an active site cleft. However, substitution of a few amino acid residues, which are conserved in the other members of the family, has been observed in both the domains and also at the region of the interdomain cleft. Consequently, the number of intra- and interdomain hydrogen-bonding interactions is enhanced in the structure of ervatamin C. Moreover, a unique disulfide bond has been identified in the right domain of the structure, in addition to the three conserved disulfide bridges present in the papain family. All these factors contribute to an increase in the stability of ervatamin C. In this enzyme, the nature of the S2 subsite, which is the primary determinant of specificity of these proteases, is similar to that of papain, but at the S3 subsite, Ala67 replaces an aromatic residue, and has the effect of eliminating sufficient hydrophobic interactions required for S3-P3 stabilization. This provides the possible explanation for the lower activity of ervatamin C toward the small substrate/inhibitor. This substitution, however, does not affect the binding of denatured natural protein substrates to the enzyme significantly, as there exist a number of additional interactions at the enzyme-substrate interface outside the active site cleft.