taxonID	type	description	language	source
8D608791DF671365FFCD2E6BFE38B484.taxon	description	For initial analyses, the Camptotheca CYP 72 A 564, CYP 72 A 565, CYP 72 A 730 and Catharanthus CYP 72 A 1 v 3 were expressed in the WAT 11 and WAT 21 yeast strains, which have been engineered to constitutively express ATR 1 or ATR 2, 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 72 A 564 and CYP 72 A 565 expressed in the WAT 11 and WAT 21 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 72 A 1 v 3 expressed in the WAT 11 and WAT 21 strains converted only loganin into secologanin and secoxyloganin (Fig. S 1, S 2, S 3). In contrast, the more divergent Camptotheca CYP 72 A 730 expressed in the WAT 11 and WAT 21 strains failed to metabolize either loganic acid or loganin (Fig. 3, S 1, S 6).	en	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: 1-13, DOI: 10.1016/j.phytochem.2020.112626, URL: http://dx.doi.org/10.1016/j.phytochem.2020.112626
8D608791DF671365FFCD2C4AFABDB246.taxon	description	For subsequent in vitro reconstitution assays, the His 6 - tagged Camptotheca CYP 72 A 564, CYP 72 A 565 and CYP 72 A 730 and Catharanthus CYP 72 A 1 v 3 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 CPR 1 full) and CPR 2 (His 6 CPR 2 full) as well as truncated soluble forms of these electron transfer proteins (His 6 CPR 1 Δ 48, His 6 CPR 2 Δ 68) were cloned into the IPTG-inducible pET 28 bacterial expression vector and purified from E. coli. Reduced carbon monoxide difference spectra (Omura and Sato, 1967) used to quantify P 450 levels indicated that all three Camptotheca His 6 - tagged CYP 72 A proteins were mixtures of properly folded protein (peak around 450 nm) with some free heme from misfolded protein (peak around 420 nm) (Fig. S 7). The calculated P 450 concentrations for these Camptotheca preparations were in the micromolar range (15.9 μM for CYP 72 A 564, 31.9 μM for CYP 72 A 565, 3.34 μM for CYP 72 A 730). Cytochrome c reduction assays (Guengerich et al., 2009) used to quantify CPR activities indicated that the full-length and truncated His 6 -- tagged CPR 1 proteins efficiently reduced cytochrome c whereas the full-length and truncated His 6 - tagged CPR 2 proteins inefficiently reduced cytochrome c at equivalent protein levels (Fig. S 8). Reconstitution of the purified His 6 - tagged CYP 72 A 564 and CYP 72 A 565 proteins with His 6 CPR 1 full and DLPC showed that both of these Camptotheca P 450 s metabolize loganic acid and loganin in the presence of NADPH (Fig. 4 A 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 P 450 s with His 6 CPR 2 full showed no detectable conversion of loganic acid or loganin (Fig. 4 C and D); reconstitutions with His 6 CPR 1 Δ 48 and His 6 CPR 2 Δ 68 showed no conversion of loganin or loganic acid (data not shown). Reconstitutions of the more divergent CYP 72 A 730 with His 6 CPR 1 full showed no conversion of loganic acid or loganin (Fig. 4 A and B, 5). Reconstitutions with full-length Catharanthu s CYP 72 A 1 v 3 showed conversion of loganin to secologanin but not conversion of loganic acid (Fig. 4 A 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 CYP 72 A 1, CYP 72 A 564, and CYP 72 A 565 reconstituted with Camptotheca His 6 CPR 1 full, no such activity was observed for CYP 72 A 730 (Supplemental Fig. S 13).	en	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: 1-13, DOI: 10.1016/j.phytochem.2020.112626, URL: http://dx.doi.org/10.1016/j.phytochem.2020.112626
8D608791DF611361FFCD2944FF78B2C5.taxon	description	Molecular models for Catharanthus CYP 72 A 1 and Camptotheca CYP 72 A 564 and CYP 72 A 565, constructed according to published procedures (Rupasinghe and Schuler, 2006), indicated that the backbones of these three proteins overlay well (RMSD less than 2.0 Å, green, Fig. 7 A) except for the following regions: the C-terminus of the A ′ - A loop, β 1 - 2, the N-terminus of the C helix, the D-E ′ loop, the N-terminus of the G-helix, the N-terminal half of the H – I loop, the middle of the loop between β 1 - 3 and β 1 - 4, and in a loop region immediately C-terminal of the PERF domain. None of these regions have RMSD values greater than 3.5 Å (orange), and all are outside the predicted SRSs. Dockings of loganin in the Catharanthus CYP 72 A 1 catalytic site and loganic acid in the Camptotheca CYP 72 A 564 and CYP 72 A 565 catalytic sites predicted that both substrates position C 10 within 4.5 Å of the oxygen bound to the heme with comparable interaction energies (Table 3); as both proposed mechanisms for the C – C bond scission demand (Guengerich and Yoshimoto, 2018; Yamamoto et al., 2000), abstraction of a hydrogen from this carbon forms the radical needed to cleave the C – C bond in loganin. Even so, the orientations of these substrates were different with loganic acid oriented more vertically in the two Camptotheca CYP 72 A proteins than loganin is in the Catharanthus CYP 72 A 1 model. Overlays of the predicted contact residues highlighting amino acids identical in each (Fig. 7 C and E) and different in each (Fig. 7 D and F) show many identical residues within substrate contact distance and relatively few different residues in this area. Among the variant residues, several differences occur in SRS 1 at positions 132, 133, 134, 135 and 138 (Fig. 2; Fig. 7 B, C, 7 E). The Catharanthus protein differs from the two Camptotheca proteins at all of these positions, and the two Camptotheca proteins differ only at position 135 (Asn / Ser). One additional difference among these three proteins occurs in SRS 3 at position 270 with all three proteins having different residues (Thr / Lys / Arg). Similar overlays of identical and different residues in these two Camptotheca proteins (Fig. 7 B – E) highlight their substantial identity in all SRSs comprising the catalytic site. Parameters ± S. E. estimated from a non-linear curve fit of corrected NADPH consumption rates against varying substrate concentrations (N = 7) from combined technical triplicate assays. Molecular modeling and docking of Camptotheca CYP 72 A 730 with loganic acid, which it neither binds nor metabolizes, suggest that this non-substrate can be positioned within the catalytic site (Fig. 8). The loganic acid in the CYP 72 A 730 model has a similar distance to the oxygen bound to the heme and interaction energy compared to the Camptotheca CYP 72 A 564 and CYP 72 A 565 models (Table 3). Overlays of the backbones in these three Camptotheca models show the most substantial (> 4.5 Å) RMSD variance extending from the F-helix through the G-helix (red in Fig. 8 A) and little in most other regions (≤ 2.0 Å, green in Fig. 8 A). Overlays of these three predicted catalytic sites highlighting amino acids identical in each (Fig. 8 C and E) and different in each (Fig. 8 D and F) show multiple residues that differ within loganic acid contact distance and within the upper regions of its predicted catalytic site. Among the variant residues, several differences occur in SRS 1: the Camptoth eca CYP 72 A 730 protein differs from the CYP 72 A 564 and CYP 72 A 565 proteins at positions 132 and 133 (numbered according to CYP 72 A 730) (Fig. 2; Fig. 8 B). Differences between these three protein models also occur in SRS 2 at position 234, SRS 3 at position 268 and SRS 4 at positions 323 and 327. Other differences among these Camptotheca proteins, while occurring in SRS regions (i. e., SRS 4 at position 325, SRS 6 at positions 499 and 501) are more than 4.5 Å away from the docked substrate. Notable among the sequence variations within substrate contact distance are those in SRS 2 at position 234 and SRS 3 at 268 (numbering according to CYP 72 A 730). Because of the substantial RMSD variance in the placement of the F-to G-helices shown in Fig. 8 A, the first of these differences (Ile 234 in CYP 72 A 730), is substantially closer to the docked loganic acid than Met 236 in CYP 72 A 564 and CYP 72 A 565. The second of these (Gln 268 in CYP 72 A 730) is displaced so significantly compared to Arg 270 in CYP 72 A 564 and Lys 270 in CYP 72 A 565 that it is not predicted to lie within substrate contact distance. The docking poses of their alternate substrate, 7 - deoxyloganic acid, in the CYP 72 A 564 and CYP 72 A 565 homology models greatly resembled those of loganic acid in these models (Supplemental Fig. S 12). Though considerable variability in the placement of the glucoside moiety is present, the iridoid cores of 7 - deoxyloganic acid and loganic acid remain nestled between SRS 1 and SRS 4. Comparison of the contacts amongst the docked substrate-P 450 pairs revealed that the residues predicted as essential for loganic acid docking (especially His 132 hydrogen bonding to the carboxylic acid) are likewise predicted contacts for 7 - deoxyloganic acid.	en	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: 1-13, DOI: 10.1016/j.phytochem.2020.112626, URL: http://dx.doi.org/10.1016/j.phytochem.2020.112626
8D608791DF6D136FFFCD2D0BFD77B080.taxon	description	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 CYP 72 A 564 and CYP 72 A 565 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 CYP 72 A 564 and CYP 72 A 565 hydroxylate geniposide, Catharanthus CYP 72 A 1 produced the same product (Supplemental Fig. S 13). 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 72 A 1 with Catharanthus CPR. With this additional 7 DLH activity confirmed for the Camptotheca SLASs, we have docked 7 - deoxyloganin in CYP 72 A 564 and CYP 72 A 565 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. S 12) 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 His 132 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 C 7 and C 10 on the iridoid scaffold, either of which are required to be in close proximity to the iron (IV) - oxo complex for hydrogen abstraction.	en	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: 1-13, DOI: 10.1016/j.phytochem.2020.112626, URL: http://dx.doi.org/10.1016/j.phytochem.2020.112626
