P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

Justin C. Miller, Allison J. Hollatz, Mary A. Schuler

Research output: Contribution to journalArticlepeer-review

Abstract

Divergent terpene indole alkaloid (TIA) pathways in Catharanthus roseus and Camptotheca acuminata generate vinblastine and vincristine, and camptothecin, respectively. In contrast to Catharanthus which feeds secologanin (from methylated loganin) into its species-specific late pathway, Camptotheca feeds secologanic acid (from unmethylated loganic acid) into its late pathway. Having identified putative Camptotheca secologanic acid synthases (SLASs) and cytochrome P450 reductases (CPRs) in transcriptome databases, we have demonstrated that two P450s, CYP72A564 and CYP72A565, are capable of utilizing both loganic acid and loganin to generate secologanic acid and secologanin. We have extended the previous report of these activities by CYP72A565 and CYP72A610 (Yang et al., 2019) by demonstrating that both Arabidopsis CPRs (ATR1, ATR2) couple with these CYP72A proteins in yeast microsomal assays and that purified Camptotheca CPR1 couples with them in in vitro reconstitution assays. Kinetic analyses of purified full-length Camptotheca SLASs have indicated that both process loganic acid with nearly identical catalytic rates and efficiencies as measured by their kcat and kcat/KM. In contrast, CYP72A564 processes loganin with two-fold greater efficiency than CYP72A565 correlating with the former's 3-fold greater affinity for loganin. The closely-related CYP72A730 does not bind or process either compound. Molecular modeling of these three proteins and comparisons with Catharanthus secologanin synthase (SLS) have identified key differences that likely determine their SLAS versus SLS selectivities. Our ability to reconstitute these SLAS/SLS activities provides valuable tools for further examinations of the residues involved in substrate recognition and determinations of their unusual mechanism of C–C bond scission.

Original languageEnglish (US)
Article number112626
JournalPhytochemistry
Volume183
DOIs
StatePublished - Mar 2021

Keywords

  • Alkaloid biosynthesis
  • Camptotheca acuminata Decne.
  • Cytochrome P450 (P450)
  • Cytochrome P450 reductase (CPR)
  • Nyssaceae
  • Plant biochemistry

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Plant Science
  • Horticulture

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