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
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Mentions: For the identification of l‐2‐haloacid dehalogenases in the HAD superfamily, two motifs could serve as primary identifiers owing to the distinct functional requirements of such enzymes (Fig. 3). At the N‐terminus, the dehalogenases harbour Asp‐X‐[Tyr/Phe] instead of the Asp‐X‐[Asp/Glu] found in phosphatases. Near the C‐terminus, the dehalogenases possess the oxyanion hole motif Ser175‐Ser176‐Asn177 (L‐Dex YL) or Ala‐Ala‐His, which contrasts with the phosphatase Mg2+‐binding pair of carboxylates ([Asp/Glu]‐Asp, Gly‐[Asp/Glu]‐X3‐Asp or Gly‐[Asp/Glu]‐X4‐Asp); the aligning residues are underlined. These two carboxylate pair motifs may be applied to reliably filter out probable phosphoryl transferases before further screening with dehalogenase‐specific residues, because they were found in numerous phosphoryl transferases (Koonin and Tatusov, 1994; Burroughs et al., 2006; Kuznetsova et al., 2006).
Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.