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: Dehalogenases catalyse the cleavage of carbon‐halogen bonds and are critical to the detoxification and biodegradation of organohalogens. Of all types of dehalogenases discovered over the past few decades (Fetzner and Lingens, 1994; Hill et al., 1999; de Jong and Dijkstra, 2003), the hydrolytic dehalogenases are particularly attractive research targets (and have thus been studied the most) because they are cytosolic proteins, generally do not require cofactors, and use water as the sole co‐substrate. A large number of hydrolytic dehalogenases belong to two large enzyme superfamilies: the α/β hydrolases (ABH; 22 133 sequences in databases, July 2009) and the haloacid dehalogenase‐like hydrolases (HAD; 25 048 sequences). The ABH superfamily includes a variety of hydrolytic enzymes with carboxylesterase, thioesterase, peptidase, haloalkane dehalogenase and fluoroacetate dehalogenase activities (Holmquist, 2000). The vast majority of HAD members have unknown function; those with known function display one of five activities: ATPase, phosphatase, phosphoglucomutase, phosphonohydrolase or l‐2‐haloacid dehalogenase (Koonin and Tatusov, 1994; Allen and Dunaway‐Mariano, 2004). Collectively, the dehalogenases from the ABH and HAD superfamilies transform a relatively broad variety of substrates, which is an advantageous property for bioremediation as well as industrial biocatalysis (Table 1) (Swanson, 1999). Interestingly, despite the absence of any sequence similarities between the ABH and HAD dehalogenases, they all employ a similar two‐step dehalogenation mechanism that involves an aspartate nucleophile and a covalent ester intermediate (Fig. 1) (Verschueren et al., 1993; Liu et al., 1998; Schmidberger et al., 2007).
Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.