Identification of a novel determinant of glutathione affinity in dichloromethane dehalogenases/glutathione S-transferases.
Bacterial dichloromethane dehalogenases catalyze the glutathione-dependent hydrolysis of dichloromethane to formaldehyde and are members of the enzyme superfamily of glutathione S-transferases involved in the detoxification of electrophilic compounds. Numerous protein engineering studies have addressed questions pertaining to the substrate specificity, the reaction mechanism, and the kinetic pathway of glutathione S-transferases. In contrast, the molecular determinants for binding of the glutathione cofactor have been less well investigated. Dichloromethane dehalogenases from Hyphomicrobium sp. DM2 and Methylobacterium sp. DM4 displayed significantly different affinities for glutathione, but not for the dichloromethane substrate. The sequence of dcmA, the dichloromethane dehalogenase gene from strain DM2, was determined and featured a single base difference from the previously determined sequence of dcmA from strain DM4. This base change resulted in a single amino acid difference in the corresponding proteins at sequence position 27. Site-directed variants of the homologous dichloromethane dehalogenase from Methylophilus sp. DM11 (56% amino acid identity) at the corresponding residue in the protein sequence provided further evidence that this residue selectively modulated the dependence of dichloromethane dehalogenase activity on glutathione.
Vuilleumier
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S
Mikrobiologisches Institut, Swiss Federal Institute of Technology, Zürich, CH-8092, Switzerland. vuilleumier@micro.biol.ethz.ch
Sorribas
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H
Leisinger
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D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D005982:Glutathione Transferase; D008190:Lyases; C045224:dichloromethane dehalogenase; D005978:Glutathione