Structural and functional studies on drosophila ADH

Abstract

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.