Camptotheca SLAS

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, Phytochemistry (112626) 183, pp. 1-13 : 4

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).

Figures

Fig. 7. Molecular models of Catharanthus CYP72A1 and Camptotheca CYP72A564 and CYP72A565. (A) Backbone overlays of Catharanthus CYP72A1 and Camptotheca CYP72A564 and CYP72A565 models are shown with the alpha-carbon RMSD amongst CYP72A1, CYP72A564 and CYP72A565 depicted from green (0.0 Å) to yellow (3.0 Å) to red (4.5 Å). (B) SRS regions in CYP72A proteins shown with predicted substrate contact residues (gray fill). (C) Identical versus (D) different side chain residues predicted within 4.5 Å of loganin (aqua) docked in Catharanthus CYP72A1 (blue) and loganic acid (gray) docked in Camptotheca CYP72A564 (orange). (E) Identical versus (F) different side chain residues predicted within 4.5 Å of loganin (aqua) docked in Catharanthus CYP72A1 (blue) and loganic acid (gray) docked in Camptotheca CYP72A565 (rose).

Fig. 8. Molecular models of Camptotheca CYP72A564, CYP72A565 and CYP72A730. (A) Backbone overlays of Camptotheca CYP72A730, CYP72A564 and CYP72A565 models are shown with the RMSD variance of CYP72A564 and CYP72A565 from the CYP72A730 backbone depicted in green (0.0 Å), yellow (3.0 Å) and red (4.5 Å). (B) SRS regions in CYP72A proteins shown with predicted substrate contact residues (gray fill). (C) Identical versus (D) different side chain residues predicted within 4.5 Å of loganic acid (gray) docked in Camptotheca CYP72A564 (orange) versus loganic acid (aqua) docked in CYP72A730 (magenta). (E) Identical versus (F) different side chain residues predicted within 4.5 Å of loganic acid (gray) docked in Camptotheca CYP72A565 (rose) versus loganic acid (aqua) docked in CYP72A730 (magenta).

Fig. 4. LC-MS analyses of in vitro assays with purified His6-tagged CYP72A proteins reconstituted with His6-tagged CPR proteins. Reactions containing purified His6- tagged CYP72A protein, full-length His6-tagged Caa CPR1 protein (A, B) or full-length His6-tagged Caa CPR2 protein (C, D), and 250 μM loganic acid (A,C) or loganin (B,D), were incubated at 30◦C and analyzed by LC-MS as described in experimental procedures. Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089); loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246) are given with stacked chromatograms as marked.

Fig. 5. Area of loganic acid, loganin and products from in vitro reconstitution assays conducted with full-length Camptotheca His6-tagged CPR1. Integrated areas from LC-MS analyses of purified His6-tagged CYP72A proteins reconstituted with full-length His6-tagged Caa CPR1 are shown for no NADPH (gray) and plus NADPH (gray slashed) reactions.