Figure 3: Expression and screening results of insect cytochrome P450s and UGTs.

(a) CO-difference spectra from yeast microsomes harbouring A, CYP405A2; B, CYP304F2; C, CYP405A3; D, CYP332A3; E, CYP9A36; F, CYP9A37; G, CYP6CT1; H, CYP6AE27; I, CYP4G47 and J, CYP4L17. (b) Screening of yeast microsome preparations for the ability of the P450s to convert Val/Ile into their corresponding oximes, 2-methylpropanal oxime (ValOx) and 2-methylbutanal oxime (IleOx) as monitored by SPME-GC-MS. Extracted ion chromatograms corresponding to the most abundant ion of the mass spectra for the two oximes (ValOx: m/z 70; IleOx: m/z 59) are shown in identical scale for all samples belonging to the respective assays. The double peaks represent the E and Z isomers of the oximes. (c) Screening of yeast microsome preparations for the ability of P450s to convert ValOx or IleOx to the corresponding cyanohydrins. Cyanohydrin formation was measured as the dissociation products acetone or 2-butanone following DNPH derivatization giving rise to acetone-DNPH and 2-butanone-DNPH monitored by LC-MS analysis. Extracted-ion chromatograms corresponding to the two adducts are shown (acetone-DNPH: m/z 239; 2-butanone-DNPH: m/z 253). CYP332A3 chromatograms have been highlighted in red. The background levels of acetone-DNPH and 2-butanone-DNPH originated from the omnipresence of acetone and 2-butanone in laboratory air and plastic ware. The capital letters in b and c represent the same P450s as in a with K being the empty vector control. (d) Screening of yeast microsomes for the ability of UGTs to convert acetone cyanohydrin (Ach) or 2-butanone cyanohydrin (Bch) into radiolabelled linamarin and lotaustralin following administration of ¹⁴C-labelled UDP-glucose, and analysis of the radiolabelled products formed by thin-layer chromatography.