TOPOLOGY OF THE MEMBRANE-BOUND ALKANE HYDROXYLASE OF PSEUDOMONAS-OLEOVORANS

摘要: The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that expressed and active in both Escherichia coli P. oleovorans. Its primary sequence contains eight hydrophobic stretches could span the as alpha-helices. topology of was studied E. using fusions linking different amino-terminal fragments (AlkB) to alkaline phosphatase (PhoA) beta-galactosidase (LacZ). Four AlkB-PhoA were constructed transposon TnphoA. Site-directed mutagenesis used create PstI sites at 12 positions AlkB. These AlkB-LacZ fusions. With respect activity each set revealed expected complementary activities. At three positions, PhoA highly active, whereas corresponding LacZ least active. all other almost completely inactive, but data predict a model for containing six transmembrane segments. In this amino terminus, two hydrophilic loops, large carboxyl-terminal domain are located cytoplasm. Only very short loops near acid 52, 112, 251 exposed periplasm.