Catalytic Site Constraints in the P450s Mediating Loganic Acid (7DLH) and Secologanic Acid Synthesis (SLAS) in Camptotheca.

Terpene indole alkaloids (TIAs) are plant-derived natural products synthesized in low levels in medicinal plants such as Catharanthus roseus and Camptotheca acuminata. TIA pathways species utilize several CYP72A subfamily members to form loganic acid from 7-deoxyloganic acid (a simple hydroxylation) as well as secologanin and secologanic acid from loganin and loganic acid (a C-C bond scission). Divergences in the specificities of these P450s have allowed Camptotheca secologanic acid synthases (SLASs) to become bifunctional enzymes capable of performing both reactions. In contrast, Catharanthus 7-deoxyloganic acid hydroxylase (7DLH) and secologanin synthase (SLS) have remained monofunctional enzymes capable either of monooxygenation or C-C bond scission. Our in vitro reconstitutions have now demonstrated that Camptotheca also contains a monofunctional 7DLH capable only of hydroxylating 7-deoxyloganic acid. Mutageneses aimed at evaluating residues important for the tight specificity of Camptotheca 7DLH (CYP72A729) and the broad specificity of SLAS (CYP72A564) have identified several residues where reciprocal switches substantially affect their activities: Lys128His in 7DLH increases hydroxylation of 7-deoxyloganic acid, and His132Lys in SLAS decreases this hydroxylation and C-C bond scissions of loganic acid and loganin; Gly321Ser in 7DLH does not affect hydroxylation of 7-deoxyloganic acid, whereas Ser324Gly in SLAS significantly increases C-C bond scission of loganic acid; Asp332Glu in the acid-alcohol pair of 7DLH increases hydroxylation of 7-deoxyloganic acid, whereas Glu335Asp in SLAS completely eliminates both of its activities. These mutations that enhance or eliminate these respective activities have significant potential to aid engineering efforts aimed at increasing TIA production in cell cultures, microbial systems, and/or other plants.

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases.

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

Engineering C-C Bond Cleavage Activity into a P450 Monooxygenase Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata.

Divergent terpene indole alkaloid (TIA) pathways in Catharanthus roseus and Camptotheca acuminata generate vinblastine and vincristine, and camptothecin, respectively. In contrast to Catharanthus which feeds secologanin (from methylated loganin) into its species-specific late pathway, Camptotheca feeds secologanic acid (from unmethylated loganic acid) into its late pathway. Having identified putative Camptotheca secologanic acid synthases (SLASs) and cytochrome P450 reductases (CPRs) in transcriptome databases, we have demonstrated that two P450s, CYP72A564 and CYP72A565, are capable of utilizing both loganic acid and loganin to generate secologanic acid and secologanin. We have extended the previous report of these activities by CYP72A565 and CYP72A610 (Yang et al., 2019) by demonstrating that both Arabidopsis CPRs (ATR1, ATR2) couple with these CYP72A proteins in yeast microsomal assays and that purified Camptotheca CPR1 couples with them in in vitro reconstitution assays. Kinetic analyses of purified full-length Camptotheca SLASs have indicated that both process loganic acid with nearly identical catalytic rates and efficiencies as measured by their kcat and kcat/KM. In contrast, CYP72A564 processes loganin with two-fold greater efficiency than CYP72A565 correlating with the former's 3-fold greater affinity for loganin. The closely-related CYP72A730 does not bind or process either compound. Molecular modeling of these three proteins and comparisons with Catharanthus secologanin synthase (SLS) have identified key differences that likely determine their SLAS versus SLS selectivities. Our ability to reconstitute these SLAS/SLS activities provides valuable tools for further examinations of the residues involved in substrate recognition and determinations of their unusual mechanism of C-C bond scission.