Generation of chimerical fatty acid desaturases with bifunctional D12/D15 regioselectivity

ALLOATTI, ANDRÉS; UTTARO, ANTONIO D.

Tucuman

Congreso; SAIB 45th Annual Meeting; 2009

Sociedad Argentina de Investigaciones Bioquímicas

We have previously characterized D12 and D15 desaturases from Leishmania major, respectively involved in conversion of oleoyl- to linoleoyl- and linoleoyl- to linolenyl-moieties of parasite´s phospholipids. Both proteins share 76% of identity and 88% of similarity. Analysis of primary structure revealed both a conserved theoretical membrane topology and consensus sequences for the three clusters of histidines, presumed to be part of the active site. However, both desaturases show some significant differences in certain regions, for example in the luminal domain located between the first and second transmembrane helixes or in some parts of the catalytic site. Construction of chimerical desaturases between both enzymes allowed us to generate hybrids that showed a bifunctional activity, catalyzing the conversion of 16:1 to 16:2, 18:1 to 18:2 and 18:2 to 18:3. On the other hand, we constructed some chimeras that totally lack desaturase activity. These results allowed us to identify structural determinants involved in substrate discrimination and (or) regioselectivity. A small N-terminal domain from D15 desaturase is sufficient to create a bifunctional enzyme when replacing the equivalent region in D12 desaturase. These findings are relevant from academic and biotechnological perspectives. This work is dedicated to the memory of Dr. Rodolfo A. UGALDE