Pre packed

Chemicals, biochemicals, buffers, solvents, and the components for Luria-Bertani (LB) media were obtained from sources reported elsewhere [11 (link)]. The synthesis of 2-hydroxymuconate (3a) [8 (link)], 2-hydroxy-2,4-pentadienoate (5a) [10 (link)], 5-chloro-2-hydroxymuconate (4Z-3b) [11 (link)], and (4Z)-5-chloro-2-hydroxy-2,4-pentadienoate (5b) [11 (link)], and ethyl 2-fluorocrotonate [18 (link)] are reported in the indicated references. The Phenyl Sepharose 6 Fast Flow resin and the pre-packed PD-10 Sephadex G-25 columns were obtained from GE Healthcare (Piscataway, NJ). The Econo-Column chromatography columns were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). 4-OT, 4-OD/VPH, and 4-OD/E106QVPH (all from P. putida mt-2) were purified by procedures reported elsewhere with minor modifications [10 (link)–11 (link)19 (link)–20 (link)]. The YM-3 ultrafiltration membranes and centrifugal microconcentrators were obtained from Millipore (Billerica, MA). Activities were determined using previously described assays [10 (link)–11 (link)19 (link)–20 (link)]. The plasmids containing the genes for 4-OT and 4-OD/VPH from P. putida mt-2 and L. cholodnii SP-6, and 4-OD/E106QVPH from P. putida mt-2 were constructed as reported elsewhere [10 (link)–11 (link)]. Proteins were expressed as described.

Echinodontium tinctorium was collected from three separate locations in north-central BC: an old growth forest near McBride (voucher not kept); Twin Falls Recreation Site, Smithers (CL37); and Pine Lake Recreation Site, Terrace (CL103). DEAE Sephadex, dextran standards (T1, T5, T12, T25, T50, T80, T150, T270, T410), and the standard monosaccharides (glucose, mannose, rhamnose, ribose, xylose, arabinose, fucose, galactose, galactosamine, and glucosamine) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was from Life Technologies Inc. (Waltham, MA, USA), and Dulbecco Modified Eagle Medium was from LONZA (Walkersville, MD, USA). Sephadex LH-20 resin and pre-packed Superdex 200 Increase 10/300 GL were from GE Healthcare (Uppsala, Sweden). Polysaccharide-K was purchased from Kureha Pharmaceuticals (Tokyo, Japan). All other reagents used were of analytical grade.