Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum.
The FAD-containing enzyme cholesterol oxidase catalyzes the oxidation and isomerization of 3beta-hydroxysteroids having a trans double bond at delta5-delta6 of the steroid ring backbone to the corresponding delta4-3-ketosteroid. Two representative enzymes of this family, namely cholesterol oxidase from Streptomyces hygroscopicus (SCO) and the recombinant enzyme from Brevibacterium sterolicum (BCO) expressed in Escherichia coli, have been characterized herein in their chemical, physical, and biochemical properties. In the native form, both enzymes are monomeric (55 kDa), acidic (pI 4.4-5.1) and contain oxidized FAD (peaks in the 370-390-nm and 440-470-nm regions). Marked differences exist between the oxidized, reduced, and (red) anion semiquinone spectra of the two enzymes, suggesting substantial differences in the flavin microenvironment. Both enzymes form reversibly flavin N(5)-sulfite adducts via measurable k(on) and k(off) steps. BCO has a higher affinity for sulfite (Kd approximately 0.14 mM) compared to SCO (approximately 24 mM). This correlates well with the midpoint redox potentials of the bound flavin, which in the case of BCO are about 100 mV more positive than for SCO. Both enzymes show a high pKa (approximately 11.0) for the N(3) position of FAD. With both enzymes, the rearrangement of 5-cholesten-3-one to 4-cholesten-3-one is not rate limiting indicating that the rate-limiting step of the overall reaction is not the isomerization. The absence of the double bond in the steroid molecule does not significantly affect turnover and affinity for the substrate, whereas both these parameters are affected by a decreasing length of the substrate C17 chain.
Gadda
G
G
Department of Structural and Functional Biology, University of Milano, Varese, Italy.
Wels
G
G
Pollegioni
L
L
Zucchelli
S
S
Ambrosius
D
D
Pilone
M S
MS
Ghisla
S
S
D001951:Brevibacterium / Q000201:enzymology*; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013447:Sulfites / Q000378:metabolism
D013447:Sulfites; D002789:Cholesterol Oxidase