Camptotheca

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 : 10

3.5. Broad substrate scope of Camptotheca SLASs

Despite their ability to modify both unmethylated and methylated derivatives of loganic acid, metabolomic data have indicated that multiple Camptotheca tissues contain loganic acid, secologanic acid and strictosidinic acid but not loganin, secologanin or strictosidine ( Sadre et al., 2016). Additional metabolomic data from Jin et al. (2019) have verified the presence of loganic acid, secologanic acid and strictosidinic acid in shoot apices and leaves but not in roots. The use of these unmethylated precursors for the formation of subsequent products represents an important bifurcation in the Camptotheca pathway from the use of methylated precursors in Catharanthus , Lonicera and many other medicinal plant species. Presuming that loganin does not exist in Camptotheca even at low levels, the ability of CYP72A564 and CYP72A565 to metabolize loganin is surprising and indicates that it has retained an ancestral activity (toward loganin) while obtaining another activity (toward loganic acid) or vice versa.

Camptotheca SLASs have also been reported to hydroxylate 7-deoxyloganic acid to form loganic acid ( Yang et al., 2019), the preceding step in their TIA pathway. Lacking access to 7-deoxyloganic acid to assess 7-hydroxylation activities of these CYPs, we used the structurally-related geniposide documented as an alternate substrate by Yang and colleagues. Not only did CYP72A564 and CYP72A565 hydroxylate geniposide, Catharanthus CYP 72A1 produced the same product (Supplemental Fig. S13). Assuming geniposide turnover is a quality surrogate for 7-deoxyloganin hydroxylation, this last result is in direct contrast to that of Irmler et al. (2000) who recorded no 7-deoxyloganin hydroxylation by an E. coli membrane fraction containing a fusion of Catharanthus CYP 72A1 with Catharanthus CPR.

With this additional 7DLH activity confirmed for the Camptotheca SLASs, we have docked 7-deoxyloganin in CYP72A564 and CYP72A565 to determine whether predicted substrate contacts for their hydroxylation activities differ from those for carbon-carbon bond scission activities on loganic acid and loganin. These dockings (Supplemental Fig. S12) show little variation in the binding poses for these various molecules near the heme. Though there are differences in contacts when comparing 7-deoxyloganic acid and loganic acid, contacts with the strongest interactions such as His132 H-bonding to the carboxylic acid moiety of both compounds remain. These similiarities in binding position and interaction are unsurprising given the close proximity of C7 and C10 on the iridoid scaffold, either of which are required to be in close proximity to the iron(IV)-oxo complex for hydrogen abstraction.