European Journal of Biochemistry 1982-10-01

Biosynthesis of ascorbate in yeast. Purification of L-galactono-1,4-lactone oxidase with properties different from mammalian L-gulonolactone oxidase.

H S Bleeg, F Christensen

Index: Eur. J. Biochem. 127(2) , 391-6, (1982)

Full Text: HTML

Abstract

An enzyme from Saccharomyces cerevisiae which catalyzes the reaction: L-galactonolactone + O2 leads to L-ascorbate + H2O2 has been purified 466-fold from the mitochondrial fraction of a yeast homogenate. The enzyme has several properties that are different from the L-galactonolactone oxidase described by Nishikimi et al. [Arch. Biochem. Biophys. 191, 479-486 (1978)]. By gel filtration in the presence of sodium deoxycholate an apparent Mr of 70 000 was obtained for the active enzyme. Polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave an Mr of 74 000, whereas sodium dodecylsulfate/polyacrylamide gel electrophoresis showed only one protein band corresponding to an Mr of 18 000. A tetrameric structure of the enzyme is thereby suggested. The substrate specificity is confined to the aldonoacid lactones L-galactono-, D-altrono-, L-fucono-, D-arabino- and D-threono-1,4-lactones. Competitive inhibition was demonstrated with L-gulono- and D-galactono-1,4-lactones. p-Chloromercuriphenyl sulfonate, iodoacetamide, N-ethylmaleimide, sulfite and sulfide were all inhibitory to the enzyme. No effect was seen when cyanide, azide, EDTA, alpha, alpha'-bipyridyl or bathocuproine disulfonate was added. An apparent Km of 0.3 mM with L-galactonolactone as a substrate was found. The Km for oxygen was 0.18 mM. The pH/activity curve exhibited a maximum around pH 8.9 and a shoulder at pH 6.5. Evidence of a covalently bound flavin coenzyme and involvement of an iron-sulfur cluster was obtained from difference spectra of oxidized minus substrate-reduced enzyme with peaks or shoulders of the oxidized enzyme at 475, 445, 410, 375 and 350 nm. In sodium dodecylsulfate/polyacrylamide gels the enzyme subunit(s) had a bright yellow fluorescence after fixation in 7% acetic acid or 5% formaldehyde. The galactonolactone oxidase is stable with 50% activity being lost in 6 months at + 5 degrees C.


Related Compounds

Related Articles:

Evolution of enzymatic activities in the enolase superfamily: L-fuconate dehydratase from Xanthomonas campestris.

2006-12-12

[Biochemistry 45(49) , 14582-97, (2006)]

Carbon-branched carbohydrate chirons: practical access to both enantiomers of 2-C-methyl-ribono-1,4-lactone and 2-C-methyl-arabinonolactone. Booth, K. V., et al.

[Tetrahedron Asymmetry 20 , 2357-2367, (2009)]

Synthesis of ·-Substituted 3-Ulosonic Acids from Aldonolactones. Csuk, R., et al.

[Tetrahedron 59 , 7887-7895, (2003)]

Biosynthesis of Vitamin B6: Origin of Pyridoxine by the Union of Two Acyclic Precursors, 1-Deoxy-D-xylulose and 4-Hydroxy-L-threonine. Kennedy, I.A., et al.

[J. Am. Chem. Soc. 117 , 1661-1662, (1995)]

More Articles...