Membrane Topology of the Acyl-Lipid Desaturase from Bacillus subtilis

DIAZ, A.R; MANSILLA, C; VILA, A.; DE MENDOZA, D

Buenos Aires

Congreso; XIV International Biophysics Congress; 2002

Fatty acid desaturases are iron-dependent enzymes that introduce doublé bonds into long chain fatty acids. Membrane fatty acid desaturases have been found in all eukaryotes, cyanobacteria and some bacilli. Structural information on these proteins is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase (AlkB). The topology of Bacillus subtilis acyl-lipid desaturase (delta5-Des), was experimentally determined in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (D5-Des-PhoA). The PhoA activities of cells expressing the D5-Des-PhoA fusions, combined with site-directed mutagenesis of conserved His residues, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data support a model for D5-Des, different from that determined of AlkB, as a polytopic membrne protein with six transmembrane and one membrane-associated domain, that likely represents a substratending motif.