Molecular cloning of a cytochrome P450 taxane 10beta -hydroxylase cDNA from Taxus and functional expression in yeast -- Data Supplement - Supplemental Data -- Proceedings of the National Academy of Sciences

Supplementary material for Schoendorf et al. (2001) Proc. Natl. Acad. Sci. USA 98 (4), 1501–1506. (10.1073/pnas.031574198)

Experimental Methods
NMR Spectrometry.
All NMR spectra were recorded on a Varian Inova-500 NMR spectrometer operating at 18°C using a very sensitive 5-mm pulsed-field gradient ¹H indirect-detection probe. The taxadien-diol monoacetate was dissolved in C₆D₆ to a final concentration of about 300 µM. 2D-TOCSY was acquired using a z-filtered DIPSI (Decoupling In the Presence of Scalar Interactions) mixing sequence, a 60-msec mixing time, 10-KHz spin-lock field, 16 repetitions, 256 (t₁) ´ 2048 (t₂) complex points, 6,500-Hz sweep in each dimension. 2D-ROESY was acquired using a z-filtered mixing sequence with a 409-msec mixing time, 4-KHz spin-lock field, 128 repetitions, 256 (t₁) ´ 2048 (t₂) complex points, 6,500-Hz sweep in each dimension. 2D-HSQC was acquired using 256 repetitions, 128 (t₁) ´ 1024 (t₂) complex points, 6,500 Hz in F2 and 15,000 Hz in F1. The time between repetitions was 1.5 sec for these experiments. Data were processed using Varian VNMR software, version 6.1C. The final data size, after linear-prediction in t₁ and zero-filling in both dimension, was 1,024(F1) ´ 2,048(F2) complex points for all experiments.