Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14±0.013 mM and 0.19±0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a Ki of 2.1±0.9 nM. A truncated BC domain that included amino acids 2–560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1±0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.
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Research Article|
May 15 2004
Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain Available to Purchase
Stephanie C. WEATHERLY;
Stephanie C. WEATHERLY
1
Cropsolution, Inc., P.O. Box 14069, Research Triangle Park, NC 27560, U.S.A.
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Sandra L. VOLRATH;
Sandra L. VOLRATH
1
Cropsolution, Inc., P.O. Box 14069, Research Triangle Park, NC 27560, U.S.A.
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Tedd D. ELICH
Tedd D. ELICH
2
Cropsolution, Inc., P.O. Box 14069, Research Triangle Park, NC 27560, U.S.A.
2To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
December 19 2003
Revision Received:
January 27 2004
Accepted:
February 09 2004
Accepted Manuscript online:
February 09 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 380 (1): 105–110.
Article history
Received:
December 19 2003
Revision Received:
January 27 2004
Accepted:
February 09 2004
Accepted Manuscript online:
February 09 2004
Citation
Stephanie C. WEATHERLY, Sandra L. VOLRATH, Tedd D. ELICH; Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain. Biochem J 15 May 2004; 380 (1): 105–110. doi: https://doi.org/10.1042/bj20031960
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