title: Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain.
abstract: 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 K(i) 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.
authors.affiliation: Cropsolution, Inc., P.O. Box 14069, Research Triangle Park, NC 27560, USA.
authors.forename: Stephanie C
authors.initials: SC
authors.lastname: Weatherly
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Volrath
authors.forename: Tedd D
authors.initials: TD
authors.lastname: Elich
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D019921:Biotinylation; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005659:Fungicides, Industrial / Q000378:metabolism*; D007700:Kinetics; D018942:Macrolides / Q000378:metabolism*; D020082:Magnaporthe / Q000201:enzymology*; D008969:Molecular Sequence Data; D010838:Phytophthora / Q000201:enzymology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013329:Structure-Activity Relationship; D014588:Ustilago / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D018942:Macrolides; D011993:Recombinant Fusion Proteins; C086151:soraphen A; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D000103:Acetyl-CoA Carboxylase