Isolation and Characterization of O-methyltransferases Involved in the Biosynthesis of Glaucine in Glaucium flavum  

Abstract

Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 49-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C69 coupling and four O-methyl groups at C6, C7, C39, and C49 as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 39 and 49 hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 49OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 49-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 39-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 39-and 79-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.

Biosynthesis of plant tetrahydroisoquinoline alkaloids through an imine reductase route

“…15 Production of racemic (R,S)-laudanosine in E. coli has been previously achieved through feeding (R,S)-norlaudanosoline to a strain expressing O-MTs and NMTs. 16 However, the poor activities of O-MTs result in low yields of (R,S)-laudanosine, and the accumulation of many methylated intermediates (e.g. (R,S)reticuline, (R,S)-codamine and (R,S)-laudanine) complicates its isolation (Fig.…”

Heterodimeric O‐methyltransferases involved in the biosynthesis of noscapine in opium poppy

“…As with other substrate‐ and regio‐specific OMTs involved in BIA metabolism, a clade of closely related OMT2 orthologs occurs in other BIA‐producing species (Hagel et al ., ) suggesting that the specific (or a related) heterodimeric activity of OMT2:OMT3 or OMT2:6OMT occurs in other plants. Previously, we surveyed seven OMT homodimers encoded by genes expressed in yellow horn poppy ( Glaucium flavum ) roots and detected four enzymes with either relatively specific or broad activities on 1‐benzylisoquinoline and protoberberine substrates (Chang et al ., ). An enzyme designated OMT4 from G .…”

“…An enzyme designated OMT4 from G . flavum exhibited no apparent activity with substrates tested, but is a close homolog of opium poppy OMT2 (Chang et al ., ; Hagel et al ., ), which suggests that OMT4 is functional only when partnered with the product of an OMT paralog. In general, the possible widespread occurrence of heterodimeric OMTs involved in BIA metabolism, and potentially beyond, could expand the diversity of substrate‐ and regio‐specific function beyond a directly proportional relationship with respect to OMT genes and metabolic conversions.…”

Survival analysis of genome-wide profiles coupled with Connectivity Map database mining to identify potential therapeutic targets for cholangiocarcinoma

“…Puromycin is a conversion inhibitor in biosynthesis and inhibits protein synthesis, and has anti-tumor, anti-parasitic and antibiotic effects ( 42 ). Cephaeline and scoulerine are associated with alkaloid synthesis ( 43 ). L-scoulerine antagonizes the dopamine D2 receptor to inhibit decreases in prepulse inhibition, which is caused by MK-801.…”