RESUMO
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. DsaA has a moderate activity level and a preference for long (greater than 3 carbons) brominated substrates, but little activity toward chlorinated alkanes. DccA shows high activity with both long brominated and chlorinated alkanes. The structure of DccA was determined by X-ray crystallography and was refined to 1.5 Å resolution. The enzyme has a large and open binding pocket with two well-defined access tunnels. A structural alignment of HLD-I subfamily members suggests a possible basis for substrate specificity is due to access tunnel size.
Assuntos
Actinobacteria/enzimologia , Caulobacter crescentus/enzimologia , Hidrolases/química , Hidrolases/metabolismo , Actinobacteria/química , Actinobacteria/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Caulobacter crescentus/química , Caulobacter crescentus/genética , Cristalografia por Raios X , Hidrolases/genética , Modelos Moleculares , Conformação Proteica , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
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. A single mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the amino acid dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates.