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 42–50kDa enzyme. Based on sequencing and expression data from the fadD gene, other reports have suggested that FadD is a 62kDa protein and represents the sole fatty acyl-CoA synthetase in E. coli. We report that the 62kDa FadD enzyme is a substrate for the outer membrane protease OmpT in vitro, producing a 43kDa C-terminal fragment and a 19kDa N-terminal fragment. Immunoblotting with a FadD antibody revealed that only the 62kDa 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 43kDa 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 Km and Vmax values compared with intact FadD, but the catalytic efficiencies (kcat/Km) 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.
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December 2001
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Research Article|
December 10 2001
Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT
Jae-Ho YOO;
Jae-Ho YOO
1
McMaster University, Hamilton, Ontario L8N 3Z5, Canada
1To whom correspondence should be addressed (e-mail jaeho@mit.edu).
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Oscar H. CHENG;
Oscar H. CHENG
McMaster University, Hamilton, Ontario L8N 3Z5, Canada
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Gerhard E. GERBER
Gerhard E. GERBER
McMaster University, Hamilton, Ontario L8N 3Z5, Canada
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Publisher: Portland Press Ltd
Received:
June 11 2001
Revision Received:
August 08 2001
Accepted:
October 04 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 360 (3): 699–706.
Article history
Received:
June 11 2001
Revision Received:
August 08 2001
Accepted:
October 04 2001
Citation
Jae-Ho YOO, Oscar H. CHENG, Gerhard E. GERBER; Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT. Biochem J 15 December 2001; 360 (3): 699–706. doi: https://doi.org/10.1042/bj3600699
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