Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members.
A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.
D000595:Amino Acid Sequence; D000818:Animals; D001379:Azathioprine / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D032621:DNA Shuffling; D018076:DNA, Complementary / Q000737:chemistry; D004274:DNA, Recombinant; D003850:Deoxyribonuclease I / Q000378:metabolism; D019020:Directed Molecular Evolution; D015723:Gene Library; D014644:Genetic Variation; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011355:Prodrugs / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013269:Stochastic Processes; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
D018076:DNA, Complementary; D004274:DNA, Recombinant; D007166:Immunosuppressive Agents; D007527:Isoenzymes; D011355:Prodrugs; D014867:Water; D005982:Glutathione Transferase; D003850:Deoxyribonuclease I; D005978:Glutathione; D001379:Azathioprine