Abstract
Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.
Highlights
Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His69 of the protein backbone
The reduced form of H69A Brevibacterium sterolicum (BCO) is spectrally different from wild-type BCO in that the 376 nm band is replaced by a shoulder at Ϸ389 nm (Fig. 1)
The present results demonstrate that covalent flavinylation is not a prerequisite for cholesterol oxidase (CO) protein folding and for efficient binding of FAD
Summary
Tains a noncovalently linked FAD [1]; the structure of the CO from Streptomyces hygroscopicus (SCO) has recently been reported [2] Both enzymes possess the classical Rossman fold for dinucleotide binding found in many flavin-dependent oxidases [3] and belong to the glucose-methanol-choline oxidoreductase family [4]. A second CO was isolated from a strain reported to be a Brevibacterium sterolicum (BCO) [5] It has a different primary sequence, it contains a FAD covalently linked to N(1) of His, and its three-dimensional structure belongs to a different family compared with the two COs mentioned above.. Comparison of the structure of the present BCO with the crystal structure of the noncovalent CO from B. sterolicum, in light of the data presented here, is expected to expand our understanding of the role of the flavin covalent attachment in flavoprotein oxidases
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