STRUCTURE OF A DEHYDRATASE-ISOMERASE FROM THE BACTERIAL PATHWAY FOR BIOSYNTHESIS OF UNSATURATED FATTY-ACIDS - 2 CATALYTIC ACTIVITIES IN ONEACTIVE-SITE

Background: Escherichia coli beta-hydroxydecanoyl thiol eater dehydras e (dehydrase) is essential to the biosynthesis of unsaturated fatty ac ids, by shunting a 10-carbon intermediate from the saturated fatty aci d pathway into the unsaturated fatty acid pathway. Dehydrase catalyzes reactions of dehydration and of double-bond isomerization on 10-carbo n thiol esters of acyl carrier protein (ACP). The aim of this work is to elucidate mechanisms for the two enzymatic reactions, which occur i n an unusual bifunctional active site, and to understand the specifici ty of the enzyme for substrates with 10-carbon fatty acyl chains. Resu lts: Crystal structures at 2.0 Angstrom resolution for free dehydrase and for the enzyme modified by its classic, mechanism-based inactivato r, 3-decynoyl-N-acetylcysteamine, have been determined. Dehydrase is a symmetric dimer with an unusual alpha + beta 'hot dog' fold. Each of the two independent active sites is located between the two subunits o f the enzyme, and is a tunnel-shaped pocket completely isolated from t he general solvent. Side chains of histidine from one subunit and aspa rtic acid from the other are the only potentially reactive protein gro ups in the active site, Conclusions: A two-base mechanism by which the histidine and aspartic acid together catalyze dehydration and isomeri zation reactions is consistent with the active-site structure. The uni que topology of the protein fold and the identification of the active- site components reveal features of predictive value for another enzyme , FabZ, which may to be the non-specific dehydratase involved in elong ation of fatty acyl chains. A positively charged area surrounding the entrance to the active site, which could interact with the negatively charged ACP, was also found.