Sequence- and activity-based screening of microbial genomes for novel dehalogenases.
Dehalogenases are environmentally important enzymes that detoxify organohalogens by cleaving their carbon-halogen bonds. Many microbial genomes harbour enzyme families containing dehalogenases, but a sequence-based identification of genuine dehalogenases with high confidence is challenging because of the low sequence conservation among these enzymes. Furthermore, these protein families harbour a rich diversity of other enzymes including esterases and phosphatases. 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. In this first biochemical screen, 1 haloalkane dehalogenase, 1 fluoroacetate dehalogenase and 5 l-2-haloacid dehalogenases were found (success rate 7%), as well as 19 esterases and 31 phosphatases. Using this functional data, we refined the sequence-based dehalogenase selection criteria and applied them to a second functional screen, which identified novel dehalogenase activity in 13 out of only 24 proteins (54%), increasing the success rate eightfold. 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.
Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
Wing Yiu
WY
Chan
Max
M
Wong
Jennifer
J
Guthrie
Alexei V
AV
Savchenko
Alexander F
AF
Yakunin
Emil F
EF
Pai
Elizabeth A
EA
Edwards
D001419:Bacteria / Q000235:genetics* / Q000378:metabolism*; D001681:Biological Assay; D019295:Computational Biology; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism*; D006219:Halogens / Q000378:metabolism*; D008403:Mass Screening / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism*
D004798:Enzymes; D006219:Halogens; D009930:Organic Chemicals
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