Purification and characterization of the recombinant form of Acyl CoA oxidase 3 from the yeast Yarrowia lipolytica.
The Acyl CoA dependent oxidase 3 (Aox3p) from the yeast Yarrowia lipolytica, expressed in Escherichia coli, as an active protein with a 6 His tag at its N-terminal region has been purified to electrophoretic homogeneity. The purified enzyme exhibits a specific activity of 1.95 microM/min/mg using hexanoyl-CoA as substrate, and it remains active for at least 1 month upon storage at -30 degrees C in the presence of 35% (V/V) glycerol. The pH and temperature optima of the enzyme are 7.4 and 28-38 degrees C, respectively. Aox3p catalyzes the oxidation of both aliphatic acyl-CoA substrates of different chain lengths (e.g., hexanoyl-CoA, decanoyl-CoA, myristyl-CoA) as well as of the aromatic/heterocyclic ring-substituted chromogenic substrates, such as furylpropionyl-CoA. Of the above substrates, the efficiency of the enzyme, as judged by its kcat to Km ratio, exhibits the following order: decanoyl CoA > myristyl CoA > hexanoyl CoA > furyl-propionyl-CoA (FPCoA). Phenol, which is normally used in the coupled assay system for monitoring the H2O2 formation, functions as both an activator (at low concentrations) and a competitive inhibitor (at high concentrations) with respect to acyl-CoA substrates. The magnitude of activation and inhibition of the enzyme is dependent on the nature of the acyl-CoA substrates.
Luo
Y S
YS
Laboratoire de Chimie Biologique, INRA-CNRS, Centre de Biotechnologie Agro-Industrielle, Thiverval-Grignon, France.
Wang
H J
HJ
Gopalan
K V
KV
Srivastava
D K
DK
Nicaud
J M
JM
Chardot
T
T
D002384:Catalysis / Q000187:drug effects; D007700:Kinetics; D010088:Oxidoreductases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D019800:Phenol / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology*
D011994:Recombinant Proteins; D019800:Phenol; D010088:Oxidoreductases; C418542:acyl CoA oxidase 3