The enzyme alcohol dehydrogenase of the fruit-fly Drosophila (DADH)
catalyzes the same reaction as the mammalian alcohol dehydrogenases,
transforming alcohols into aldehydes through the reduction of nicotinamideadenine-dinucleotide. Despite this identical chemical behaviour the enzyme's
structure belongs to a different class of proteins, called short-chain
dehydrogenases, which have a totally different three-dimensional architecture
to the mammalian alcohol dehydrogenases. The study of the structure-fuction
relationships of DADH is of interest because, while we still lack a crystal
structure for the protein, large amounts of biochemicafevolutionary and
genetical data have accumulated which require structural information on the
enzyme for its proper interpretation.
The aim of this project was two-fold: to set up a suitable system for the
undertaking of protein engineering studies on DADH and to start such studies
by producing and analyzing a first set of site-directed mutants. The first part
of the project involved: a) creating suitable genetic vectors for the introduction
of mutations, their sequencing and the expression of the mutated enzymes in
yeast; b) the development of a purification method to obtain pure enzyme
solutions from the expressing yeast culture; c) the development of biochemical
and biophysical assays for the evaluation of the mutation's effects and, d) the
construction of a three-dimensional model for the enzyme that could offer
structural explanations to such effects.
The second part consisted of the actual introduction of five different
mutations in the enzyme's sequence, and their further evaluation using the
system set up in the first place.