ABSTRACT
Acyl-CoA oxidases (ACOXs) play important roles in lipid metabolism, including peroxisomal fatty acid ß-oxidation by the conversion of acyl-CoAs to 2-trans-enoyl-CoAs. The yeast Yarrowia lipolytica can utilize fatty acids as a carbon source and thus has extensive biotechnological applications. The crystal structure of ACOX3 from Y. lipolytica (YlACOX3) was determined at a resolution of 2.5 Å. It contained two molecules per asymmetric unit, and the monomeric structure was folded into four domains; Nα, Nß, Cα1, and Cα2 domains. The cofactor flavin adenine dinucleotide was bound in the dimer interface. The substrate-binding pocket was located near the cofactor, and formed at the interface between the Nα, Nß, and Cα1 domains. Comparisons with other ACOX structures provided structural insights into how YlACOX has a substrate preference for short-chain acyl-CoA. In addition, the structure of YlACOX3 was compared with those of medium- and long-chain ACOXs, and the structural basis for their differences in substrate specificity was discussed.
Subject(s)
Acyl-CoA Oxidase/chemistry , Acyl-CoA Oxidase/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Yarrowia/enzymology , Acyl Coenzyme A/metabolism , Acyl-CoA Oxidase/genetics , Acyl-CoA Oxidase/isolation & purification , Amino Acid Sequence , Animals , Biotechnology , Caenorhabditis elegans/enzymology , Catalytic Domain , Cloning, Molecular , Coenzymes/chemistry , Crystallography, X-Ray , Escherichia coli/genetics , Fatty Acids/metabolism , Flavin-Adenine Dinucleotide , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Gene Expression Regulation, Fungal , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/isolation & purification , Protein Conformation , Protein Domains , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Yarrowia/geneticsABSTRACT
N-ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.
