Drosophila lebanonensis ADH: analysis of recombinant wild-type enzyme and site-directed mutants. The effect of restoring the consensus sequence in two positions.
Unique amino acid substitutions occur in D. lebanonensis ADH. They are found within the putative NAD(+)-binding domain and affect residues that are otherwise highly conserved in all other species of the genus. To restore the consensus amino acids, we have constructed an expression system for this enzyme in E. coli, and engineered two mutants, Ala13Gly and Asn56Thr. The biochemical and kinetic features of these retromutants are consistent with increased catalytic efficiency and thermal stability. Thus, results show that wild-type D. lebanonensis ADH can be improved by site-directed mutagenesis.
Albalat
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R
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Spain.
Atrian
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S
Gonzàlez-Duarte
R
R
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D004330:Drosophila / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase