CHARACTERIZATION OF CHOLESTEROL OXIDASE FROM STREPTOMYCES-HYGROSCOPICUS AND BREVIBACTERIUM-STEROLICUM

The FAD-containing enzyme cholesterol oxidase catalyzes the oxidation and isomerization of 3 beta-hydroxysteroids having a trans double bond at Delta(5)-Delta(6) Of the steroid ring backbone to the correspondin g Delta(4)-ketosteroid. Two representative enzymes of this family, nam ely cholesterol oxidase from Streptomyces hygroscopicus (SCO) and the recombinant enzyme from Brevibacterium sterolicum (BCO) expressed in E scherichia coli, have been characterized herein in their chemical, phy sical, and biochemical properties. In the native form, both enzymes ar e monomeric (55 kDa), acidic (pi 4.4-5.1) and contain oxidized FAD (pe aks in the 370-390-nm and 440-470-nm regions). Marked differences exis t between the oxidized, reduced, and (red) anion semiquinone spectra o f the two enzymes, suggesting substantial differences in the flavin mi croenvironment. Both enzymes form reversibly flavin N(5)-sulfite adduc ts via measurable k(on) and k(off) steps. BCO has a higher affinity fo r sulfite (K-d approximate to 0.14 mM) compared to SCO (approximate to 24 mM). This correlates well with the midpoint redox potentials of th e bound flavin, which in the case of BCO are about 100 mV more positiv e than for SCO. Both enzymes show a high pK(a) (approximate to 11.0) f or 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 isomeri zation. The absence of the double bond in the steroid molecule does no t significantly affect turnover and affinity for the substrate, wherea s both these parameters are affected by a decreasing length of the sub strate C17 chain.