A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase

Abstract

Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3beta-hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH...pi interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall k(cat). Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0 A resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH...pi interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered.

Figure 1

Catalytic mechanism of cholesterol oxidase.

Figure 2

Electron density of cholesterol oxidase active site. 2F _o − F _c electron density (blue mesh), contoured at 1.0σ, is shown for (a) the N485L mutant and (b) the N485D mutant. Active-site residues and the FAD cofactor are shown as ball-and-stick models. Alternate conformations of side chains have been labeled A and B.

Figure 3

Comparison of cholesterol oxidase active sites. (a) WT enzyme, (b) N485L mutant, (c) N485D mutant. Only the isoalloxazine portion of FAD is shown for clarity. Hydrogen bonds are shown as dashed lines. Water molecules are labeled W. Alternate conformations for side chains are labeled A and B.

Figure 4

Effect of mutation on the putative oxygen-access channel. (a)–(f) Space-filling representations of the solvent-accessible surface in the vicinity of the cholesterol oxidase active site. (a, c, e) Active-site residues in conformation A, with the channel closed. (b, d, f) Active-site residues in conformation B, with the channel open in the WT enzyme. Mobile residues of the (a, b) WT enzyme, (c, d) the N485L mutant and (e, f) the N485D mutant are shown in ball-and-stick representation.

Figure 5

A model of the unusual hydrogen-bonding interactions of Asp485 with Glu361 and Met122. (a) Conformation A, with Glu361 OE2 protonated and negative charge localized on Asp485 OD2; (b) conformation B, with Glu361 not protonated and negative charge delocalized on both Glu361 and Asn485. Note that in both cases the methyl of Met122 interacts with negatively charged moieties.