The acyl-CoA oxidases from the yeast Yarrowia lipolytica: characterization of Aox2p.
One of the acyl-CoA oxidases from the yeast Yarrowia lipolytica, acyl-CoA oxidase 2 (Aox2p), has been expressed in Escherichia coli as an active, N-terminally tagged (His)(6) fusion protein. The specific activity of the purified enzyme, containing FAD, was 19.7 micromolmin(-1)mg(-1) using myristoyl-CoA as substrate. Using substrates with different chain lengths and different substituents, its kinetic properties were further analyzed. Straight-chain acyl-CoAs, with a chain length of 10-14C, are well oxidized, reflecting the properties of Aox2p as deduced from in vivo studies. Acyl-CoAs containing more than 14C were also desaturated, if their concentration was below 25 microM or if proteins capable of binding these CoA-esters, such as albumin or beta-casein, were added to the assay. These long-chain acyl-CoAs, although poor substrates, acted as competitors for the short- and medium-chain substrates. Compared to palmitoyl-CoA, activity toward hexadecadioyl-CoA, containing a omega-carboxy group, was similar. Taken together, these data suggest that micelles of long-chain acyl-CoAs are able to bind and inhibit Aox2p. The enzyme was also active toward acyl-CoA-esters containing a 2-methyl group, but only the 2S isomer was recognized.
Luo
Yi Shan
YS
Laboratoire de Chimie Biologique, INRA INA-PG, Centre de Biotechnologie Agro-Industrielle, Thiverval-Grignon, France.
Nicaud
Jean Marc
JM
Van Veldhoven
Paul P
PP
Chardot
Thierry
T
D000214:Acyl Coenzyme A / Q000378:metabolism; D042962:Acyl-CoA Oxidase; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D025062:Yarrowia / Q000201:enzymology*; D000099188:Alternative Oxidase
D000214:Acyl Coenzyme A; D042962:Acyl-CoA Oxidase; D005182:Flavin-Adenine Dinucleotide; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099188:Alternative Oxidase; C040349:S-tetradecanoyl-coenzyme A