CYP96T1 of Narcissus sp. aff. pseudonarcissus Catalyzes Formation of the Para-Para' C-C Phenol Couple in the Amaryllidaceae Alkaloids

Abstract

The Amaryllidaceae alkaloids are a family of amino acid derived alkaloids with many biological activities; examples include haemanthamine, haemanthidine, galanthamine, lycorine, and maritidine. Central to the biosynthesis of the majority of these alkaloids is a C-C phenol-coupling reaction that can have para-para', para-ortho', or ortho-para' regiospecificity. Through comparative transcriptomics of Narcissus sp. aff. pseudonarcissus, Galanthus sp., and Galanthus elwesii we have identified a para-para' C-C phenol coupling cytochrome P450, CYP96T1, capable of forming the products (10bR,4aS)-noroxomaritidine and (10bS,4aR)-noroxomaritidine from 4'-O-methylnorbelladine. CYP96T1 was also shown to catalyzed formation of the para-ortho' phenol coupled product, N-demethylnarwedine, as less than 1% of the total product. CYP96T1 co-expresses with the previously characterized norbelladine 4'-O-methyltransferase. The discovery of CYP96T1 is of special interest because it catalyzes the first major branch in Amaryllidaceae alkaloid biosynthesis. CYP96T1 is also the first phenol-coupling enzyme characterized from a monocot.

Keywords: Amaryllidaceae alkaloids; cytochrome P450; phenol coupling; secondary metabolism; transcriptomics.

Figure 1

Proposed biosynthetic pathways for representative Amaryllidaceae alkaloids directly derived from C-C phenol coupling. The previously discovered NpN4OMT, the CYP96T1 discovered in this study, and potential enzyme classes involved in each step of the pathways are in blue.

Figure 2

Structures of relevant compounds.

Figure 3

Work-flow for identification of candidate cytochrome P450 enzymes. Following the generation of transcriptome assemblies, cytochrome P450 enzymes were identified with BLASTP (Navy blue) and genes correlating with N4OMT were identified with HAYSTACK (Red). The genes present in both lists makeup the initial candidate gene list (Green). Homologs of these genes were identified in the N4OMT correlating lists of the other transcriptomes using BLASTN (Gray). Candidates with homologs in all five N4OMT correlating lists were cloned from N. sp. aff. pseudonarcissus, Narcissus sp. (light blue). The analysis for the N. sp. aff. pseudonarcissus ABySS and MIRA assembly is completely diagrammed to illustrate the process followed in every assembly. The number of transcripts selected in each step is in parentheses. The N. sp. aff. pseudonarcissus Trinity assembly is excluded from this work-flow due to its poor quality.

Figure 4

MUSCLE alignment of protein sequences. The sequences include CYP96T1, CYP96T2, CYP96T3, the CYP96T1 from the N. sp. aff. pseudonarcissus ABySS and MIRA assembly, and CYP96A15 from A. thaliana (Q9FVS9). Simplified consensus motifs for cytochrome P450 enzymes are placed above the corresponding color inverted CYP96T1 sequence. Dots are exact matches to CYP96T1 and dashes are gaps.

Figure 5

LC-MS/MS enhanced product ion scan (EPI) monitoring the C-C phenol coupling of 4′-O-methylnorbelladine and 4′-O-methyl-N-methylnorbelladine in CYP96T1 assays. Arrows indicate peaks unique to Sf9 cell containing assays with substrate present. (A) Standards and assays with 4′-O-methylnorbelladine as the substrate. Sample runs top to bottom (10bR,4aS)- and (10bS,4aR)-noroxomaritidine standard (1 μM), CYP96T1 assay, CPR assay, assay without Sf9 cells, and CYP96T1 assay without 4′-O-methylnorbelladine. (B) Standards and assays with 4′-O-methyl-N-methylnorbelladine as the substrate. Top to bottom narwedine standard, CYP96T1 assay, CPR assay, assay without Sf9 cells, and assay without 4′-O-methylnorbelladine. (C) EPI of the (10bR,4aS)- and (10bS,4aR)-noroxomaritidine standard. (D) EPI of the CYP96T1 (10bR,4aS)- and (10bS,4aR)-noroxomaritidine product with 4′-O-methylnorbelladine as substrate. (E) EPI of the CYP96T1 para-para' product (Unknown 1) with 4′-O-methyl-N-methylnorbelladine as substrate. Red fragments indicate the addition of one methyl group, 14 m/z, relative to (10bR,4aS)- and (10bS,4aR)-noroxomaritidine and blue fragments indicate the same m/z as (10bR,4aS)- and (10bS,4aR)-noroxomaritidine fragments. Intensity is presented in counts per second (CPS).

Figure 6

Chromatographic separation and MS/MS analysis of the primary 4′-O-methylnorbelladine products (10bS,4aR)- and (10bR,4aS)-noroxomaritidine. The enantiomers (10bS,4aR)- and (10bR,4aS)-noroxomaritidine were chromatographically separated with a chiral-CBH column and analyzed by MS/MS using an enhanced product ion (EPI) scan. (A) Samples, top to bottom: (10bR,4aS)- and (10bS,4aR)-noroxomaritidine standard, CYP96T1 assay, CPR assay, CYP96T1 assay without 4′-O-methylnorbelladine substrate and no Sf9 cells assay. (B) EPI fragmentation pattern for enantiomer 1 of (10bR,4aS)- and (10bS,4aR)-noroxomaritidine. (C) EPI fragmentation pattern for enantiomer 2 of (10bR,4aS)- and (10bS,4aR)-noroxomaritidine. (D) EPI fragmentation pattern for enantiomer 1 in the CYP96T1 assay with 4′-O-methylnorbelladine as substrate. (E) EPI fragmentation pattern for enantiomer 2 in the CYP96T1 assay with 4′-O-methylnorbelladine as substrate. Intensity is presented in counts per second (CPS).

Figure 7

Relative product formed in assays with 4′-O-methylnorbelladine (A,B) or 4′-O-methyl-N-methylnorbelladine (C,D,E) as substrate. Assays are performed in triplicate only expressing CPR or with CPR in combination with CYP96T1. (A) para-para' [(10bR,4aS)- and (10bS,4aR)-noroxomaritidine] product. (B) para-ortho' (N-demethylnarwedine) product. (C) Potentially para-para' C-C phenol coupling (unknown 1) product. (D) para-ortho' (Narwedine) product. (E) Potentially ortho-para' C-C phenol coupling (unknown 2) product.

Figure 8

LC-MS/MS Enhanced Product Ion (EPI) scan of sodium borohydride (NaBH₄) treated CYP96T1 assays with 4′-O-methylnorbelladine substrate. (A) Chromatograph with the following sample runs top to bottom: N-demethylgalanthamine standard, CYP96T1 assay, CPR assay, assay with no Sf9 cells and CYP96T1 assay without 4'-O-methylnorbelladine. (B) EPI fragmentation pattern of the N-demethylgalanthamine standard peak eluting at 4 min. (C) EPI fragmentation pattern of the N-demethylgalanthamine product in the CYP96T1 assay. (D) EPI fragmentation pattern of epi-N-demethylgalanthamine from the CYP96T1 assay. (E) EPI fragmentation pattern of (10bS,4aR)- and (10bR,4aS)-noroxomaritidine standard reduced to stereoisomeric 8-O-demethylmaritidine. (F) EPI fragmentation pattern of reduced (10bS,4aR)- and (10bR,4aS)-noroxomaritidine product from CYP96T1 assays.

Figure 9

Proposed C-C phenol coupling mechanisms. (A) 4′-O-methylnorbelladine para-para' C-C phenol coupling mechanism followed by spontaneous nitrogen ring closure to form noroxomaritidine. (B) (R)-reticuline para-ortho' C-C phenol coupling mechanism to form salutaridine panel adapted from Grobe et al. (2009).