Isolation and characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in sacred lotus (Nelumbo nucifera)

Abstract

Benzylisoquinoline alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value because of antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline structural subclasses and displaying a predominant R-enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing >40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R- and S-substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S-enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-benzylisoquinoline intermediates. K_m values for NnOMT1 and NnOMT5 were 20 and 13 μm for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.

Keywords: Nelumbo nucifera; O-methyltransferases; benzylisoquinoline alkaloids; enzyme catalysis; mass spectrometry (MS); natural product; natural product biosynthesis; plant biochemistry; plant molecular biology; regiospecificity; secondary metabolism; stereospecific.

Figure 1.

Schematic representation of the proposed 1-benzylisoquinoline alkaloid biosynthetic pathway in sacred lotus (N. nucifera). The enzymes functionally characterized in this study (NnOMT1 and NnOMT5) are indicated, and detected alkaloids are shown in bold. Asterisk denotes the chiral center in norcoclaurine. Gray arrow, norcoclaurine synthase (NCS); green arrows, 6-O-methyltransferase (6OMT); red arrows, 7-O-methyltransferase (7OMT); blue arrows, 4′-O-methyltransferase (4′OMT); black arrows, N-methyltransferase (NMT).

Figure 2.

(a) Diagram of sacred lotus. (b) Heat map showing the organ-specific profile of BIAs in two varieties of sacred lotus (Pink and White). Relative abundance corresponds to the mean value of three independent replicates. Values were normalized to the sample with the highest level for each compound. (c) Structures corresponding to detected aporphine, pro-aporphine, and bisbenzylisoquinoline alkaloids isolated from sacred lotus (for 1-benzylisoquinoline alkaloid structures refer to Fig. 1). FL, folded leaf; UL, unfolded leaf; Rh, rhizome; Ro, roots; E, embryos.

Figure 3.

Phylogenetic relationships among sacred lotus O-methyltransferase (NnOMT) candidates and functionally characterized OMTs from BIA-accumulating plants in the Ranunculales. The evolutionary history was inferred using the Maximum Likelihood and the JTT matrix-based model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree with the highest log likelihood is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGAX. Cc, C. chinensis; Cj, C. japonica; Ct, C. teeta; Ec, E. californica; Gf, G. flavum; Nn, N. nucifera; Ps, P. somniferum; Tf, T. flavum; Tt, Thalictrum tuberosum. Sacred lotus NnOMT1 (green box) and NnOMT5 (red box) are indicated.

Figure 4.

Multiple sequence alignment among NnOMT candidates and T. flavum 6OMT (Tf6OMT). Residues shared by Tf6OMT and sacred lotus OMTs are shaded in gray, and fully conserved residues are shaded in black. Asterisks indicate catalytic (His²⁵⁶, Asp²⁵⁷, and Glu³¹⁵) and other key residues implicated in BIA (Gly¹⁶⁵, Asp¹⁶⁹, Cys²⁵³, and Asp³⁰⁶) and SAM (Thr¹⁷⁰, Gly¹⁹⁵, Asp²¹⁸, Asp²³⁸, and Lys²⁵²) binding, as reported for Tf6OMT. Conserved motives I–IV are underlined.

Figure 5.

Substrate range for recombinant NnOMT1 and NnOMT5. Values represent the mean ± S.D. of three independent replicates. The structure of the corresponding substrates is shown; nd, not detected.

Figure 6.

Organ-specific correlational analysis of 1-benzylisoquinoline alkaloids metabolism in two varieties (Pink and White) of sacred lotus. (a) Content of selected 1-benzylisoquinoline alkaloids. (b) 6OMT, 7OMT, and 4′OMT specific activity of plant crude extracts when incubated with (R,S)-norcoclaurine, (S)-N-methylcoclaurine, and (R)-armepavine, respectively. (c) Relative abundance of NnOMT1-NnOMT5 transcripts. Values represent the mean ± S.D. of three independent measurements. FL, folded leaf; UL, unfolded leaf; Rh, rhizome; Ro, roots; E, embryos.