CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice
Article first published online: 17 NOV 2010
© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd
The Plant Journal
Volume 65, Issue 1, pages 87–95, January 2011
How to Cite
Wang, Q., Hillwig, M. L. and Peters, R. J. (2011), CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice. The Plant Journal, 65: 87–95. doi: 10.1111/j.1365-313X.2010.04408.x
- Issue published online: 22 DEC 2010
- Article first published online: 17 NOV 2010
- Accepted manuscript online: 19 OCT 2010 11:30PM EST
- Received 15 August 2010; revised 5 October 2010; accepted 11 October 2010; published online 17 November 2010.
Figure S1. Western blots of CYP99A2, and CYP99A3, as indicated, following expression in Sf21 insect cell cultures as 6xHis tagged proteins, and as detected with an anti-His tag probe.
Figure S2. Optimized gene sequence for CYP99A2.
Figure S3. Optimized gene sequence for CYP99A3.
Figure S4. Summary of NMR structural analyses. (a) Carbon numbering for the syn-pimara-7(8),15(16)-dien-ol, al, or oic acid and syn-stemoden-19-ol,al,or oic acid diterpenoids. (b) Observed HMBC NMR spectra signals for syn-pimara-7(8),15(16)-dien-ol and syn-stemoden-19-ol. These are representative of the aldehydes and acids as well, minus the C19 proton correlations since C19 in the aldehydes and acids is quaternary. (c) Important cross-peak signals from NOESY spectra for the alcohols were used to determine which C4 geminal methyl group, C18 or C19, was the oxidized carbon. There was a strong signal between the alcohol protons and the C20 methyl group (known to be (R) beta from the syn configuration) protons for syn-pimara-7(8),15(16)-dien-ol and syn-stemoden-19-ol, indicating that C19 ((R) or beta on C4) is the oxidized carbon.
Figure S5. Kinetic plots for rCYP99A3 with (a) syn-pimaradiene and (b) syn-stemodene.
Figure S6. Detection of syn-pimaradien-19-oic acid, but not syn-stemoden-19-oic acid, from rice plant metabolites (both analyzed as methyl ester derivatives). Extracted GC-MS chromatogram of authentic standard, methyl jasmonate induced fractionated rice leaf extract (72 h time point), and uninduced (control) fractionated rice leaf extract for (a) syn-pimaradien-19-oic acid (m/z = 241), and (b) syn-stemoden-19-oic acid (m/z = 257).
Table S1.1H and 13C NMR assignments for syn-pimara-7,15-dien-19-ol (500 MHz at 25°C in chloroform-d).
Table S2.1H and 13C NMR assignments for syn-pimara-7,15-dien-19-al (700 MHz at 25°C in chloroform-d).
Table S3.1H and 13C NMR assignments for syn-pimara-7,15-dien-19-oic acid (500 MHz at 25°C in chloroform-d).
Table S4.1H and 13C NMR assignments for syn-stemod-13(17)-en-19-ol (500 MHz at 25°C in methanol-d4).
Table S5.1H and 13C NMR assignments for syn- stemod-13(17)-en-19-al (500 MHz at 25°C in methanol-d4).
Table S6.1H and 13C NMR assignments for syn- stemod-13(17)-en-19-oic acid (500 MHz at 25°C in methanol-d4).
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