Background: The anticancer drug camptothecin (CPT), first isolated fromCamptotheca acuminata, was subsequently discovered in unrelated plants, includingOphiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT inC. acuminatais biosynthesized via the key intermediate strictosidinic acid, but howO. pumilasynthesizes CPT has not been determined.Results: In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist inO. pumila. After identifying the enzymesOpLAMT,OpSLS, andOpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants,C. acuminataandNothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway inO. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis.Conclusions: In our study, we found thatO. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs fromC. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis inO. pumila,C. acuminata, andN. nimmonianahave evolved divergently. Overall, our new data regarding CPT biosynthesis inO. pumilasuggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.

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.

Celastrol, isolated from the traditional Chinese medicinal herb Tripterygium wilfordii Hook. f. (Thunder God's Vine), has been used to treat cancer, chronic inflammatory, autoimmune and other human diseases. However, to date, the protein targets and the mechanism of action of celastrol have remained elusive. In this study, we find that celastrol can react with protein thiols in a unique covalent and reversible manner, while protein denaturing disrupts the interaction. Through a competitive chemoproteomics approach utilizing a cysteine-targeting activity-based probe, we report the proteome-wide quantitative profiling of cellular targets of celastrol in human cervical cancer HeLa cells. Representative targets are further validated via in vitro binding experiments and/or enzymatic activity assays. Bioinformatics analysis results suggest that celastrol exerts its numerous therapeutic effects through interaction with promiscuous proteins involved in various biological processes and cellular pathways.

Rifamycin-derived drugs, including rifampin, rifabutin, rifapentine, and rifaximin, have long been used as first-line therapies for the treatment of tuberculosis and other deadly infections. However, the late steps leading to the biosynthesis of the industrially important rifamycin SV and B remain largely unknown. Here, we characterize a network of reactions underlying the biosynthesis of rifamycin SV, S, L, O, and B. The two-subunit transketolase Rif15 and the cytochrome P450 enzyme Rif16 are found to mediate, respectively, a unique C–O bond formation in rifamycin L and an atypical P450 ester-to-ether transformation from rifamycin L to B. Both reactions showcase interesting chemistries for these two widespread and well-studied enzyme families.