A heme-containing ascorbate oxidase from Pleurotus ostreatus.
A novel type of ascorbate oxidase was purified 420-fold from the cytosolic fraction of the mycelia of Pleurotus ostreatus with an overall yield of 13%. The molecular mass of the native enzyme determined by high performance gel permeation chromatography was 94 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme consists of two subunits with a molecular mass of 46 kDa. The N-terminal amino acid sequence of the enzyme was Asp-Val-Lys-Thr-Leu-Gln-Glu-His-Leu-Gln-Leu-Ala-Leu-Met-Val-. The enzyme was optimally active at pH 5.2, monitored at 37 degrees C. The enzyme had affinity toward L-ascorbic acid, D-ascorbic acid, L-erythroascorbic acid, and D-erythroascorbic acid. Under optimal conditions, the Km value of the enzyme toward L-ascorbic acid was 0.48 mm. The absorption spectra of the native enzyme exhibited a Soret maximum at 418 nm in its oxidized form and at 426 nm in its reduced form, and alpha and beta bands at 558 and 527 nm only in its reduced form, respectively. On the basis of spectral changes after treatment with cyanide and carbon monoxide, the enzyme is a hemoprotein, quite similar to b-type cytochrome, and contains 2 mol of heme per molecule. The reaction catalyzed by the enzyme was L-ascorbic acid + O2 --> dehydro-L-ascorbic acid + H2O2.
Kim
Y R
YR
Laboratory of Biophysics, Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, Republic of Korea.
Yu
S W
SW
Lee
S R
SR
Hwang
Y Y
YY
Kang
S O
SO
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001204:Ascorbate Oxidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006418:Heme / Q000032:analysis*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011124:Polyporaceae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity; D013816:Thermodynamics; D013098:Xylariales / Q000201:enzymology
D000596:Amino Acids; D010446:Peptide Fragments; D006418:Heme; D001204:Ascorbate Oxidase