Extended Data Fig. 7: Characterization of GsOMT4.
From: Discovery and engineering of colchicine alkaloid biosynthesis
a, Addition of GsOMT4 into the N. benthamiana transient co-expression system with co-infiltrated 1 leads to consumption of 8 (m/z 372.1805) and the production of a new compound corresponding to a methylation (m/z 386.1962), as shown by the LC–MS chromatograms. Comparison to an O-methylandrocymbine (9) standard purified from C. autumnale plants supports the identity of this compound as 9. This result was confirmed in more than three independent experiments. b, MS/MS fragmentation spectrum of the generated m/z 386.1962 product (*) compared to the purified 9 standard, with both compounds fragmented at a collision energy of 20 V. This was performed twice, with similar results observed each time. c, Tabulated list and putative structures for ion fragments from the MS/MS analysis of the m/z 386.1962 product. d, Untargeted metabolite analysis (XCMS) comparing the presence and absence of GsOMT4 in the transient co-expression system (n = 6 independent replicates for each experimental condition). The unique mass signatures (P < 0.1 between samples, as determined by XCMS) are shown in ranked order based on their increasing (top) or decreasing (bottom) fold change in abundance between the two conditions. The mass signature for the product (9, m/z 386.1962) is shown in red; the mass signature of the presumed substrate (m/z 372.1805) is shown in blue. e, Proposed reaction catalysed by GsOMT4, as supported by MS/MS fragmentation, previously published labelling studies and comparison to an isolated 9 standard.
