Expression and purification of his-tagged rat peroxisomal acyl-CoA oxidase I wild-type and E421 mutant proteins.
Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned the gene of rat peroxisomal acyl-CoA oxidase I into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal-affinity column in 90% yield to apparent homogeneity. The specific activity of the purified His-tagged rat peroxisomal acyl-CoA oxidase I was 1.5 micromol/min/mg. It has been proposed that Glu421 is a catalytic residue responsible for deprotonation of alpha-proton of acyl-CoA substrate. We constructed four mutant expression plasmids of the enzyme, pACO(E421D), pACO(E421A), pACO(E421Q), and pACO(E421G) using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal-affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Glu421 is a catalytic residue of rat peroxisomal acyl-CoA oxidase I. Our overexpression in E. coli and one-step purification of the highly active N-terminal His-tagged rat peroxisomal acyl-CoA oxidase I greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by peroxisomal acyl-CoA oxidase I.
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China.
Jia
J
Zeng
Ding
D
Li
D042962:Acyl-CoA Oxidase; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids; D051381:Rats
D006639:Histidine; D010088:Oxidoreductases; C107176:peroxisomal acyl-CoA oxidase; D042962:Acyl-CoA Oxidase