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Drosophila lebanonensis ADH: analysis of recombinant wild‐type enzyme and site‐directed mutants
Author(s) -
Albalat Ricard,
Atrian Sílvia,
Gonzàlez-Duarte Roser
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)80451-6
Subject(s) - mutant , site directed mutagenesis , mutagenesis , amino acid , biochemistry , enzyme , wild type , recombinant dna , biology , nad+ kinase , binding site , directed evolution , chemistry , gene
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 ainino acids, we have constructed an expression system for this enzyme in E. coli , and engineered two mutants, Ala 13 Gly and Asn 56 Thr. 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.