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

2.4. In vitro analysis of Camptotheca SLAS activities in Saccharomyces cerevisiae

For initial analyses, the Camptotheca CYP 72A564, CYP72A565, CYP72A730 and Catharanthus CYP 72A1v3 were expressed in the WAT11 and WAT21 yeast strains, which have been engineered to constitutively express ATR1 or ATR2, respectively ( Urban et al., 1997). HPLC analyses of in vitro assays conducted with yeast microsomes followed by LC-MS analyses indicated that, not unexpectedly, both Camptotheca CYP 72A564 and CYP72A565 expressed in the WAT11 and WAT21 yeast strains converted loganic acid into secologanic acid ( Fig. 3 , S 1 , S 4 , S 5 ). In addition, both of these Camptotheca enzymes also converted loganin into secologanin (detected as its sodium salt). Catharanthus CYP 72A1v3 expressed in the WAT11 and WAT21 strains converted only loganin into secologanin and secoxyloganin ( Fig. S1 , S 2 , S 3 ). In contrast, the more divergent Camptotheca CYP 72A730 expressed in the WAT11 and WAT21 strains failed to metabolize either loganic acid or loganin ( Fig. 3 , S 1 , S 6 ).

Figures

Fig. 3. LC-MS analyses of in vitro assays with CYP72A proteins expressed in yeast microsomes. 100 μl reactions containing 10 μl CYP72A microsomes isolated from WAT11 with 250 μM loganic acid or loganin and 500 μM NADPH in 100 mM NaPO (pH 7.4) were incubated at 30◦C and analyzed by LC-MS as 4 described in the experimental procedures. (A) Extracted ion chromatograms for loganic acid (m/z 375.1297), secologanic acid (m/z 373.1140), secoxyloganic acid (m/z 389.1089). (B) Extracted ion chromatograms for loganin sodium salt (m/z +413.1418), secologanin sodium salt (m/z +411.1262), secoxyloganin (m/z 403.1246).

Fig. 1. Proposed divergence of the TIA pathway between Camptotheca and Catharanthus. After 7-deoxyloganic acid hydroxylase (7DLH) converts 7-deoxyloganic acid to loganic acid, the pathways in these two species diverge. The Catharanthus pathway uses loganic acid methyltransferase (LAMT) to convert loganic acid into loganin and secologanin synthase (SLS) to convert loganin into secologanin. The Camptotheca pathway bypasses LAMT and uses secologanic acid synthase (SLAS) to metabolize loganic acid directly to secologanic acid.

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.

Fig. 2. CYP72A multiple sequence alignment. Signal anchor domain fusion of CYP72A565 into CYP72A564 is underlined; SRS regions are underlined in bold; predicted substrate contacts within 4.5 Å of loganic acid/loganin are gray-filled.

Fig. 6. Type I binding spectra of SLAS candidates. Substrate-induced Type I binding spectra for (A) CYP72A564, (B) CYP72A565 and from 998 nM (violet) to 1.90 mM (crimson) using loganic acid (top inset) or loganin (bottom inset). (C) Binding isotherms calculated as the difference of the valley (~419 nm) from the peak (~388 nm) are shown for loganic acid () and loganin (). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)