Membrane topology of the acyl-lipid desaturase from Bacillus subtilis

MANSILLA, M C; DIAZ, A R; VILA, AJ; DE MENDOZA, D

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2002 vol. 277 p. 48099 - 48106

0021-9258

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.