Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Heterologous Expression of Recombinant CYP710A Proteins in Insect Cells. SDS-PAGE analysis of the recombinantly expressed P450 proteins. Lane 1, microsomes of mock-infected Sf9 cells; lane 2, microsomes of Sf9 cells infected with the recombinant virus containing the CYP710A1 coding sequence; lane 3, microsomes of Sf9 cells infected with the recombinant virus containing the CYP710A2 coding sequence; lane 4, microsomes of Sf9 cells infected with the recombinant virus containing CYP710A cDNA. Arrows indicate the positions of the recombinantly expressed P450 proteins. The positions of molecular mass markers are shown at the left.
• Supplemental Figure 3 - Multiple sequence alignment used for phylogenetic analysis visualized using CINEMA5 (Parry-Smith et al., 1998, http://umber.sbs.man.ac.uk/dbbrowser/CINEMA2.1/). CYP710-related sequences included CYP710A1 (At 710A1) and CYP710A2 (At 710A2) from Arabidopsis, CYP710A11 (Se 710A11) from tomato, CYP710A (Mt 710A) from M. truncatula, CYP710A5 (Os 710A5) from O. sativa, CYP710B (Chlmy 710B) from C. reinhardtii, CYP710B (Cynd 710B) from C. merolae, CYP524 (Dict 524) from D. discoideum, CYP61 (pombe 61) from S. pombe, CYP61 (NC 61) from N. crassa, CYP61 (CA 61) fromC. albicans, CYP61 (Sc 61) from S. cerevisiae, CYP61 (Asprg 61) from A. fumigatus, CYP61 (Um 61) from U. maydis, CYP51F1 (Asprg 51) from A. nidulans, CYP51F1 (Sc 51) from S. cerevisiae, CYP51G1 (At 51G) from Arabidopsis, and CYP51B1 (Myc 51) from M. tuberculosis. Major Δ²²-sterols from higher plants (stigmasterol, brassicasterol, and crinosterol), C. reinhardtii (ergosterol), and D. discoideum (dictyosterol) are shown next to the corresponding genes. Polar positive residues (H, K, R) are in blue, polar negative residues (D, E) are in red, polar neutral residues (S, T, N, Q) are in green, non-polar aliphatic residues (A, V, L, I, M) are in white, non-polar aromatic (F, Y, W) are in purple, P and G are in brown , and C is shown with yellow.
• Supplemental Figure 2 - GCMS in the selected ion-monitoring mode (SIM) for CYP710A11 assay. A, stigmasterol; B, campesterol; C, crinosterol; D, 24-epi-campesterol (containing 30% campesterol as impurity); E, crinosterol produced from the contaminated campesterol in the synthesized 24-epi-campesterol (D). The m/z value of 394 was used for SIM analysis of stigmasterol production, and m/z = 380 was used for the detection of brassicasterol/crinosterol.