Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

DIAZ, ALEJANDRA R., MANSILLA, MARIA C., VILA, ALEJANDRO J., AND DE MENDOZA, DIEGO

JOURNAL OF BIOLOGICAL CHEMISTRY (ONLINE)

The American Society for Biochemistry and Molecular Biology

Año: 2002 vol. 277 p. 48099 - 48106

1083-351X

The Bacillus subtilis acyl-lipid desaturase (5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of 5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of 5-Des with the reporter alkaline phosphatase (5-Des-PhoA). The alkaline phosphatase activities of cells expressing the 5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for 5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.