PCR-Based Cloning of Membrane-Bound Desaturases

Abstract

The degree of unsaturation of fatty acids in membrane and reserve lipids is controlled by the action of different desaturase enzymes. Apart from scientific motivation, the prospects of genetic manipulation of plants have raised additional interest in the cloning of desaturases, particularly in view of the possibility to design plant oils of specifically adjusted unsaturation. In the last years several membrane-bound desaturases from plants and cyanobacteria were cloned by strategies based on complementation of mutants and protein purification. Comparison of the deduced amino acid sequences revealed three regions of homology with the general sequence of HXXXH that are highly conserved in membrane-bound desaturases from plants, cyanobacteria, yeast and mammals. These boxes may provide metal-chelating ligands contributing to the binding of oxygen in the reaction center. Oxygen functions as final acceptor of electrons derived from reduced cytochrome b₅ or ferredoxin and the ethylene segment of an acyl chain to be desaturated. These histidine boxes can be used to search for additional or even new desaturases from various organisms. First results of this approach are presented in the following.