Coenzyme a

HCT enzyme assays towards 4-coumaroyl CoA (“forward reaction”) were perform with 10 or 20 ng of recombinant proteins or 4 to 7 µg of crude plant protein extracts in a reaction solution of 100 mM sodium phosphate buffer pH 7.5, 500 µM shikimic acid and 500 µM dithiothreitol (Roche, Madison, WI) in a final volume of 100 µL. The 4-coumaroyl CoA concentration was 50 µM for assays with crude extracts, and varied from 5 to 100 µM to determine kinetics of recombinant enzymes. HCT enzyme assays towards caffeoyl shikimate (“reverse reaction”) were carried out with 50 ng of recombinant proteins or 13 to 17 µg of plant crude protein extracts in a reaction solution of 100 mM sodium phosphate buffer pH 7.5, 500 µM Coenzyme A and 500 µM dithiothreitol (Roche, Madison, WI) in a total volume of 100 µL. The caffeoyl shikimate concentration was 50 µM for assays with crude extracts, and varied from 20 to 400 µM to determine kinetics of recombinant enzymes. Reactions were terminated by addition of 10 µL of glacial acetic acid and products were analyzed by HPLC as previously described (Escamilla-Trevino et al. [27 ]) Products were quantified by measuring peak areas and converting to units of quantity using calibration curves that were constructed with authentic standards of each product.