Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea.

Two putative haloalkane dehalogenases (HLDs) of the HLD-I subfamily, DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea, have been identified based on sequence comparisons with functionally characterized HLD enzymes. The two genes were synthesized, functionally expressed in E. coli and shown to have activity toward a panel of haloalkane substrates.

Alteration of substrate specificity of alanine dehydrogenase.

The l-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: it is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for l-alanine over all other substrates. By contrast, engineering of the amino acid dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH.