FIG. 5.
Evaluation of stereospecificity of enoyl-CoA hydratase encoded by phaJAc. (a) Hydration of crotonyl-CoA coupled with (S)-specific dehydrogenation of 3HB-CoA catalyzed by (S)-3HA-CoA dehydrogenase. The reaction mixture was composed of 0.25 mM crotonyl-CoA, 0.5 mM NAD+, 6 mU of (S)-3HA-CoA dehydrogenase, and 1 U of hydratase in 400 μl of 50 mM Tris-HCl (pH 8.0). (b) Hydration of crotonyl-CoA coupled with (R)-specific polymerization of 3HB-CoA catalyzed by crude PHA synthase. The reaction mixture was composed of 0.25 mM crotonyl-CoA, 10 mM DTNB, 1.2 mU of PHA synthase (a crude extract of A. eutrophus H16 containing 10 μg of protein), and 1 U of hydratase in 400 μl of 125 mM potassium phosphate (pH 7.2). The crude extract of A. eutrophus H16 was prepared from cells grown on fructose for 30 h at 30°C, as described previously (25). Symbols: ○, addition of phaJAc-derived enoyl-CoA hydratase; □, addition of crotonase [(S)-specific enoyl-CoA hydratase]; ▴, no addition of enoyl-CoA hydratase.