H. Xiang et al., Interchange of catalytic activity within the 2-enoyl-coenzyme a hydratase isomerase superfamily based on a common active site template, BIOCHEM, 38(24), 1999, pp. 7638-7652
The structures and chemical pathways associated with the members of the 2-e
noyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a
common active site design provides the members of this family with a CoA b
inding site, an expandable acyl binding pocket, an oxyanion hole for bindin
g/polarizing the thioester C=O, and multiple active site stations for the p
ositioning of acidic and basic amino acid side chains for use in proton shu
ttling. It is hypothesized that this active sire template can be tailored t
o catalyze a wide range of chemical transformations through strategic posit
ioning of acid/base residues among the active site stations. To test this h
ypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isome
rase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed
mutagenesis to include the two glutamate residues functioning in acid/base
catalysis in a second family member, crotonase. Catalysis of the syn hydra
tion of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogen
ase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA
dehalogenase at k(cat) = 0.06 s(-1) and K-m = 50 mu M.