Camptotheca SLAS
Creators: name: Miller, Justin C.
name: Hollatz, Allison J.
name: Schuler, Mary A.
Publisher: name: Zenodo
Year: 2021
Subjects: Biodiversity
Taxonomy
Plantae
Tracheophyta
Magnoliopsida
Cornales
Nyssaceae
Camptotheca

description: 2.5. In vitro reconstitution of Camptotheca SLAS activities   
 For subsequent in vitro reconstitution assays, the His 6 -tagged Camptotheca CYP 72A564, CYP72A565 and CYP72A730 and Catharanthus CYP 72A1v3 ORFs were cloned into the IPTG-inducible pCW bacterial expression vector and their full-length proteins were purified from E. coli as outlined in the experimental procedures. His 6 -tagged full-length membrane-bound forms of Camptotheca CPR 1 (His 6 CPR1full) and CPR2 (His 6 CPR2full) as well as truncated soluble forms of these electron transfer proteins (His 6 CPR1Δ48, His 6 CPR2Δ68) were cloned into the IPTG-inducible pET28 bacterial expression vector and purified from E. coli. Reduced carbon monoxide difference spectra (Omura and Sato, 1967) used to quantify P450 levels indicated that all three Camptotheca His 6 -tagged CYP72A proteins were mixtures of properly folded protein (peak around 450 nm) with some free heme from misfolded protein (peak around 420 nm) (Fig. S7). The calculated P450 concentrations for these Camptotheca preparations were in the micromolar range (15.9 μM for CYP72A564, 31.9 μM for CYP72A565, 3.34 μM for CYP72A730). Cytochrome c reduction assays (Guengerich et al., 2009) used to quantify CPR activities indicated that the full-length and truncated His 6 --tagged CPR1 proteins efficiently reduced cytochrome c whereas the full-length and truncated His 6 -tagged CPR2 proteins inefficiently reduced cytochrome c at equivalent protein levels (Fig. S8).   
 Reconstitution of the purified His 6 -tagged CYP72A564 and CYP72A565 proteins with His 6 CPR1full and DLPC showed that both of these Camptotheca P450s metabolize loganic acid and loganin in the presence of NADPH (Fig. 4A and B, 5). For both proteins, products from loganic acid include significant amounts of secologanic acid and smaller amounts of secoxyloganic acid—an overoxidation of the aldehyde to a carboxylic acid. Both proteins also produced secologanin from loganin. Similar reconstitutions of these two Camptotheca P450s with His 6 CPR2full showed no detectable conversion of loganic acid or loganin (Fig. 4C and D); reconstitutions with His 6 CPR1Δ48 and His 6 CPR2Δ68 showed no conversion of loganin or loganic acid (data not shown). Reconstitutions of the more divergent CYP72A730 with His 6 CPR1full showed no conversion of loganic acid or loganin (Fig. 4A and B, 5). Reconstitutions with full-length Catharanthu s CYP72A1v3 showed conversion of loganin to secologanin but not conversion of loganic acid (Fig. 4A and B, 5).   
 Lacking access to the 7-deoxyloganic acid needed to assess the 7-hydroxylation activity of these CYPs reported by Yang et al. (2019), we assayed the structurally-related geniposide listed as an alternate substrate. Although we observed product formation consistent with geniposide hydroxylation for CYP72A1, CYP72A564, and CYP72A565 reconstituted with Camptotheca His 6 CPR1full, no such activity was observed for CYP72A730 (Supplemental Fig. S13).
description: Published as part of Miller, Justin C., Hollatz, Allison J. & Schuler, Mary A., 2021, P 450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata, pp. 1-13 in Phytochemistry (112626) (112626) 183 on page 4, DOI: 10.1016/j.phytochem.2020.112626, http://zenodo.org/record/8291734