Acyl-CoA oxidase from Candida tropicalis

Abstract

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.

Two acyl-coenzyme A oxidases in peroxisomes of the yeast Candida tropicalis: primary structures deduced from genomic DNA sequence.

“…Other methods have been described (10,15 (12). The subunit of the enzyme was about 75,000 in molecular weight, consistent with observations by other workers (12,20), and was indistinguishable from PXP-5, which was expected to have a homology with PXP-4 (10). It is unlikely that PXP-4 is a precursor of acyl-CoA oxidase, because oleic acid induced more PXP-4 relative to PXP-5 than n-alkanes with shorter chain lengths (Clr-C12) did.…”

Controlling Electron Transfer in Acyl-CoA Oxidases and Dehydrogenases

“…The latter is consistent with previous findings that acyl-CoA oxidase from Candida tropicalis shows an unusual thioesterase activity with acetoacetyl thioester-type ligands, although no thioesterase activity could be detected in the presence of acyl-CoA oxidase natural substrates (44). A mixture of acyl-Co oxidase isozymes purified from the organism C. tropicalis was used for this study (45). Thioesterase activity has not been observed for the ACD family.…”

A Sulfhydryl Oxidase from Chicken Egg White

“…For the measurement of cysteine plus half-cystine content, the oxidase (2 M) was denatured in 5.9 M guanidine hydrochloride, incubated with 1 mM DTT for 2 h, and alkylated with 4 mM iodoacetamide for 3 h. Excess reagents were removed by dialysis against two changes of distilled water, and the alkylated protein was lyophilized prior to amino acid analysis. Tryptophan content was estimated spectrophotometrically using the procedure of Edelhoch (23,24). The oxidase was denatured in 6 M guanidine hydrochloride, and the resulting visible spectrum was compared with an equivalent amount of free FAD in denaturant (24).…”

“…Tryptophan content was estimated spectrophotometrically using the procedure of Edelhoch (23,24). The oxidase was denatured in 6 M guanidine hydrochloride, and the resulting visible spectrum was compared with an equivalent amount of free FAD in denaturant (24). The difference spectrum, representing the contribution of the apoprotein, gave 16 tryptophan residues/subunit.…”