Mutation of Tyr375 to Lys375 allows medium-chain acyl-CoA dehydrogenase to acquire acyl-CoA oxidase activity.
Medium-chain acyl-CoA dehydrogenase (MCAD) and acyl-CoA oxidase (ACO) are key enzymes catalyzing the rate-determining step for the beta-oxidation of fatty acids. Tyr375 of MCAD is conserved in all acyl-CoA dehydrogenases and is an important residue for substrate binding. Four Tyr375 variant enzymes of rat liver MCAD were obtained through site-directed mutagenesis. Y375K was found to have intrinsic acyl-CoA oxidase activity, which was confirmed using HPLC analysis, while the wild-type and other Tyr375 variant enzymes did not show detectable oxidase activity. The kinetic parameters for the oxidase activity of Y375K variant enzyme were determined to be k(cat) of 320+/-80 h(-1) and K(M) of 30+/-15 microM using hexanoyl-CoA as the substrate. The oxidase activity of Y375K increased more than 200 times compared with that reported for the MCAD wild-type enzyme from mammalian sources. Molecular modeling study shows that the solvent accessible area for Y375K variant enzyme is wider than that of the wild-type enzyme, which indicates that Tyr375 may function as a switch against solvent accession. The mutation of this residue to Lys375 allows molecular oxygen to enter into the catalytic site serving as the electron acceptor for the reduced FAD cofactor.
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China; Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha 410083, PR China.
Jia
J
Zeng
Yuandong
Y
Liu
Long
L
Wu
Ding
D
Li
D042964:Acyl-CoA Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D042962:Acyl-CoA Oxidase / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D003067:Coenzymes / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000235:genetics*; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D051381:Rats; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics*
D003067:Coenzymes; D050505:Mutant Proteins; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D042962:Acyl-CoA Oxidase; D042964:Acyl-CoA Dehydrogenase; D008239:Lysine