Characterization of benzylisoquinoline alkaloid methyltransferases in Liriodendron chinense provides insights into the phylogenic basis of angiosperm alkaloid diversity

Abstract

SUMMARY Benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites with great pharmacological value. Their biosynthetic pathways have been extensively elucidated in the species from the Ranunculales order, such as poppy and Coptis japonica, in which methylation events play central roles and are directly responsible for BIA chemodiversity. Here, we combined BIA quantitative profiling and transcriptomic analyses to identify novel BIA methyltransferases (MTs) from Liriodendron chinense, a basal angiosperm plant. We identified an N‐methyltransferase (LcNMT1) and two O‐methyltransferases (LcOMT1 and LcOMT3), and characterized their biochemical functions in vitro. LcNMT1 methylates (S)‐coclaurine to produce mono‐ and dimethylated products. Mutagenesis experiments revealed that a single‐residue alteration is sufficient to change its substrate selectivity. LcOMT1 methylates (S)‐norcoclaurine at the C6 site and LcOMT3 methylates (S)‐coclaurine at the C7 site, respectively. Two key residues of LcOMT3, A115 and T301, are identified as important contributors to its catalytic activity. Compared with Ranunculales‐derived NMTs, Magnoliales‐derived NMTs were less abundant and had narrower substrate specificity, indicating that NMT expansion has contributed substantially to BIA chemodiversity in angiosperms, particularly in Ranunculales species. In summary, we not only characterized three novel enzymes that could be useful in the biosynthetic production of valuable BIAs but also shed light on the molecular origin of BIAs during angiosperm evolution.

Identification and Characterization of Glycosyltransferases Involved in the Biosynthesis of Neodiosmin

“…Further analysis was carried out by our previous HPLC methods. 41 HPLC analysis of the reaction products was carried out by LC-2030 Plus (Shimadzu Corporation, Tokyo, Japan) with a ZORBAX SB-C18 column (Agilent, 5.0 μm, 4.6 mm × 250 mm). The HPLC analysis method was consistent with our previous study.…”

“…The HPLC analysis method was consistent with our previous study. 41 MS analysis was performed with an LTQ-Orbitrap-XL (Thermo Fisher, USA) mass spectrometer. A Thermo Fisher UHPLC−MS/MS system and a TSQ Quantum Ultra triple-quadrupole mass spectrometer (Thermo Fisher Scientific, USA) were fitted with an ESI source.…”

“…Dried samples of each tissue were weighed 100 mg separately and extracted by sonication with 1 mL of 70% methanol (v/v) in an ultrasonic bath for 30 min twice. Further analysis was carried out by our previous HPLC methods …”

“…HPLC analysis of the reaction products was carried out by LC-2030 Plus (Shimadzu Corporation, Tokyo, Japan) with a ZORBAX SB-C18 column (Agilent, 5.0 μm, 4.6 mm × 250 mm). The HPLC analysis method was consistent with our previous study . MS analysis was performed with an LTQ-Orbitrap-XL (Thermo Fisher, USA) mass spectrometer.…”

Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera

“…Dozens of OMTs involved in the BIA biosynthetic pathway have been isolated from several species in the order Ranunculales [ 6 ] . Recently, seven OMTs from Corydalis yanhusuo and two OMTs from Liriodendron belonging to the order Magnoliales were functionally characterized [ 7 , 8 ]. These OMTs primarily catalyzed the methylation of three BIA scaffolds: 1-benzylisoquinolines, protoberberine, and phthalideisoquinolines, including norcoclaurine 6- O -methyltransferase (6OMT), 3′-hydroxy- N -methylcoclaurine 4′- O -methyltransferase (4′OMT), reticuline 7-O-methyltransferase (7OMT), norreticuline 7- O -methyltransferase (N7OMT), scoulerine-9- O -methyltransferase (SOMT), scoulerine 2- O -methyltransferase (S2OMT), columbamine O -methyltransferase (CoOMT), and 4′- O -desmethyl-3- O -acetylpapaveroxine 4′- O -methyltransferase (PsOMT2:PsOMT3 and PsOMT2:Ps6OMT heterodimers) [ 9 – 17 ].…”