Fig. 2: Promiscuous methyltransferase catalyses psi-ionone to α-irone.
From: Total enzymatic synthesis of cis-α-irone from a simple carbon source
a Cell lysates overexpressing IspD (negative control) or pMT1 were used to assay against psi-ionone. The reaction mixtures were incubated at 28 °C for 2 days and subjected to headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME-GCMS) analysis. Methylated products that correspond to trans-α-irone and cis-α-irone were detected in the reaction containing pMT1. The retention time and mass spectrum of the irones detected are the same as the synthetic chemical standard (std) from Sigma Aldrich. b Cis-α-irone, trans-α-irone and β-irone were quantified for each pMT mutant by HS-SPME-GCMS against an external standard curve. The bar chart represents the average fold change in cis-α-irone concentration compared to pMT1. The ratio between cis-α-irone to trans-α-irone concentration was calculated and represented as orange squares. The percentage of cis-α-irone in the irone mixture was calculated by dividing cis-α-irone concentration by the total irone concentration and represented by blue diamonds. The average and the standard deviation (s.d.) of two biologically independent experiments are shown. c Crystallographic structure of hexameric pMT1 in complex with a cofactor S-adenosylhomocysteine (yellow sticks) and the substrate teleocidin A1 (pink sticks) (PDB id: 5GM2). Enlarged view of amino acid residues lining the active site is shown. Three-dimensional models of pMT1 with d cis-α-irone (1S5R and 1R5S in magenta) e trans-α-irone (1S5S and 1R5R in green) are shown. pMT1 is shown as cartoon with one chain in cyan and the second one in grey. The 24 amino acid positions selected for site mutagenesis are displayed as orange sticks. Labels of residues from the second chain are underlined. Source data are provided as a Source Data file. The pdb files are available as Supplementary data 1.
