Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase.
Tyr152 and Lys156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short-chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr152 to Glu or Gln, Lys156 to Ile, Gly184 to Leu, and the double mutant Gly130 to Cys and Gly133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr152 and Lys156 are involved in catalysis and that Gly130, Gly133 and Gly184 contribute substantially to the structure of the active form.
Cols
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Department de Genètica, Facultat de Biologia, Universitat de Barcelona, Spain.
Marfany
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G
Atrian
S
S
Gonzàlez-Duarte
R
R
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014443:Tyrosine / Q000235:genetics
D014443:Tyrosine; D000426:Alcohol Dehydrogenase; D008239:Lysine