Protein engineering studies of dichloromethane dehalogenase/glutathione S-transferase from Methylophilus sp. strain DM11. Ser12 but not Tyr6 is required for enzyme activity.
The structural gene for dichloromethane dehalogenase/glutathione S-transferase (GST, EC 2.5.1.18) from Methylophilus sp. strain DM11 was subcloned into a multicopy plasmid under the control of the T7 polymerase promoter, allowing expression in Escherichia coli and easy purification of the enzyme in good yield. Several point mutations leading to amino acid changes at residues Tyr6, His8 and Ser12 of the protein were introduced in this gene. Mutations at Tyr6, the N-terminal tyrosine known to be essential for enzymatic activity in glutathione S-transferases of the alpha, mu, and pi classes, had little effect on the activity of dichloromethane dehalogenase. The same applied for mutations at residue His8, which from multiple alignments of GST sequences may also correspond to the conserved N-terminal tyrosine residue of GST enzymes. The higher turnover rate of the wild-type enzyme with dibromomethane compared with dichloromethane was lost in mutants with amino acid replacements at residue His8, but retained in mutant proteins at Tyr6. Mutations at Ser12 led to mutants with drastically reduced enzymatic activity, pinpointing this residue as an essential determinant of catalytic efficiency.
Vuilleumier
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S
Mikrobiologisches Institut, ETH Zürich, Switzerland.
Leisinger
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T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D017931:DNA Primers; D005399:Fishes; D005798:Genes, Bacterial; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008190:Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014443:Tyrosine
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D012694:Serine; D005982:Glutathione Transferase; D008190:Lyases; C045224:dichloromethane dehalogenase