Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT.
Several studies have described FadD, the Escherichia coli fatty acyl-CoA synthetase [also known as fatty acid:CoA ligase (AMP-forming); EC 6.2.1.3], as a 42-50 kDa enzyme. Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62 kDa protein and represents the sole fatty acyl-CoA synthetase in E. coli. We report that the 62 kDa FadD enzyme is a substrate for the outer membrane protease OmpT in vitro, producing a 43 kDa C-terminal fragment and a 19 kDa N-terminal fragment. Immunoblotting with a FadD antibody revealed that only the 62 kDa form of the enzyme is present in vivo, but we utilized the proteolytic sensitivity of FadD to investigate its structure. Photoaffinity labelling experiments revealed that both intact FadD and the 43 kDa fragment bound a long-chain fatty acid. Intact and cleaved FadD were also purified to determine the effect of cleavage on function. When using oleate as a substrate, cleaved FadD displayed 2-fold higher K(m) and V(max) values compared with intact FadD, but the catalytic efficiencies (k(cat)/K(m)) of the two forms were similar. This indicated that cleavage did not adversely affect enzyme activity. Proteolysis of FadD by OmpT was altered by the presence of oleate or ATP, both of which are ligands for the fatty acyl-CoA synthetase. This suggested that FadD undergoes ligand-induced conformational changes and implies that the region surrounding the cleavage site is mobile, a common characteristic of linker domains.
Yoo
J H
JH
McMaster University, Hamilton, Ontario L8N 3Z5, Canada. jaeho@mit.edu
Cheng
O H
OH
Gerber
G E
GE
D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D044943:Fatty Acid Desaturases; D005822:Genetic Vectors; D007700:Kinetics; D008970:Molecular Weight; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*
D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D044943:Fatty Acid Desaturases; C084083:Fad7 protein, Arabidopsis; D044942:Acyl-CoA Dehydrogenase, Long-Chain; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease; D003066:Coenzyme A Ligases