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-tra nsferase (GST, EC 2.5.1.18) from Methylophilus sp. strain DM11 was sub cloned into a multicopy plasmid under the control of the T7 polymerase promoter, allowing expression in Escherichia coli and easy purificati on of the enzyme in good yield. Several point mutations leading to ami no acid changes at residues Tyr6, His8 and Ser12 of the protein were i ntroduced in this gene. Mutations at Tyr6, the N-terminal tyrosine kno wn to be essential for enzymatic activity in glutathione S-transferase s of the alpha, mu and pi classes, had little effect on the activity o f dichloromethane dehalogenase. The same applied for mutations at resi due His8, which from multiple alignments of GST sequences may also cor respond to the conserved N-terminal tyrosine residue of GST enzymes. T he higher turnover rate of the wild-type enzyme with dibromomethane co mpared with dichloromethane was lost in mutants with amino acid replac ements at residue His8, but retained in mutant proteins at Tyr6. Mutat ions at Ser12 led to mutants with drastically reduced enzymatic activi ty, pinpointing this residue as an essential determinant of catalytic efficiency.