Sequence- and activity-based screening of microbial genomes for novel dehalogenases.
Bottom Line: Reliable sequence determinants are necessary to harness genome sequencing-efforts for accelerating the discovery of novel dehalogenases with improved or modified activities.In an attempt to extract dehalogenase sequence fingerprints, 103 uncharacterized potential dehalogenase candidates belonging to the α/β hydrolase (ABH) and haloacid dehalogenase-like hydrolase (HAD) superfamilies were screened for dehalogenase, esterase and phosphatase activity.Four new L-2-haloacid dehalogenases from the HAD superfamily were found to hydrolyse fluoroacetate, an activity never previously ascribed to enzymes in this superfamily.
Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.Show MeSH
Mentions: Applying the Asp nucleophile and either pair of halideâbinding residues (Trp and Trp/Asn) to filter the initial list of ABH sequences would have narrowed the list to only four of the initial 110 candidates as true potential haloalkane dehalogenases (Fig.â4; only 61 sequences are shown because they correspond to highâyield and/or active protein samples), increasing the chances of finding SAV4779 to 1 in 4. However, the absence of dehalogenase activity in the remaining three (which all carry the halideâbinding Trp pair of type I haloalkane dehalogenases) indicates that these features alone are not sufficient to pinpoint haloalkane dehalogenases. While the search may be refined by incorporating residues that line the active site or serve as supplementary halide binders, the low sequence conservation and the possibility of misalignment of true structural equivalents of the more diverse ABH proteins may lead to the dismissal of genuine dehalogenases. Nevertheless, the haloalkane dehalogenase search space could already be greatly reduced just by considering these catalytic residues. The criteria may be further customized to vary their stringency, such as by accommodating functional mutations as demonstrated in (Kennes etâal., 1995; Damborsky etâal., 1998).
Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.