DROSOPHILA-LEBANONENSIS ADH - ANALYSIS OF RECOMBINANT WILD-TYPE ENZYME AND SITE-DIRECTED MUTANTS - THE EFFECT OF RESTORING THE CONSENSUS SEQUENCE IN 2 POSITIONS

Unique amino acid substitutions occur in D. lebanonensis ADH. They are found within the putative NAD+-binding domain and affect residues tha t 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, Ala13Gl y and Asn56Thr. The biochemical and kinetic features of these retromut ants are consistent with increased catalytic efficiency and thermal st ability. Thus, results show that wild-type D. lebanonensis ADH can be improved by site-directed mutagenesis.