Cholesterol oxidases are bifunctional flavoenzymes that catalyze
two reactions in one active site. The first reaction is oxidation
of cholesterol to cholest-5-en-3-one and the second is isomerization
to cholest-4-en-3-one (Scheme 1). Streptomyces sp. SA-COO and
Rhodococcus equi cholesterol oxidases each contain the FAD cofactor
non-covalently bound to the enzyme. Their crystallographic models
reveal structurally conserved active-sites. They are members of
the GMC (glucose-methanol-choline) oxidoreductase family of flavoenzymes
in which two residues, His447 and Asn485 thought to be involved
in substrate oxidation are semi-conserved (8).
Scheme 1
A structure of the complex of the reduced enzyme with dehydro-epi-andro-sterone
showed the steroid bound in a deeply buried active site with the
hydroxyl group near to a bound water molecule (Wat541) (9). The
position of this water molecule in the active site originally
suggested that deprotonation of the steroid hydroxyl proton was
mediated by His447 through the bound water molecule (9). We recently
refined the X-ray structure of the Streptomyces enzyme to sub-Ångstrom
resolution (0.95 Å).5 This much more detailed view of the
active site has led us to propose a new model for the Michaelis
complex of the enzyme (Figure 1) and to reinterpret the mechanism
of general base catalysis (5). In order for efficient hydride
transfer to occur, the C-H bond of the substrate must be aligned
with the lowest unoccupied molecular orbital (LUMO) of the FAD,
a p-type orbital on N5 of the cofactor. Positioning the substrate
so as to maximize orbital overlap places the hydroxyl group at
the position of the bound water molecule (Wat541) in both the
native structure (5,10,11) and the substrate/reduced enzyme structure
(9). This new model for the Michaelis complex suggests, therefore,
that the bound water molecule in the native enzyme structure mimics
the substrate hydroxyl group.
Figure 1: Raster3D (3) presentation of active-site
resi-dues His447, Glu361 and Asn485, and substrate ana-log dehydroepiandrosterone,
AND, in the active site of type I cholesterol oxidase. The dehydroepi-andro-ster-one
has been modeled in the substrate binding position proposed by
Lario et al (5).
Sequence and structure alignments of the active site revealed
His447 is completely conserved within the GMC oxidoreductase family
(9) and mutagenesis and kinetic studies implied its importance
in cholesterol oxidation (10,12). The homologous histidine residues
in family members glucose oxidase (13) and cellobiose dehydrogenase
(14) are also critical for oxidation of their respective substrates.
In addition, in the case of glucose oxidase, the protonated histidine
provides the optimal protein dielectric for electron transfer
to O2 (15) In cholesterol oxidase, this residue was proposed to
act as the base for abstraction of the hydroxyl hydrogen atom
of the steroid substrate. However, the atomic resolution crystal
structure of cholesterol oxidase revealed that His447 is present
as the neutral imidazole with a hydrogen atom on NE2 of His447,
the nitrogen atom oriented towards the substrate hydroxyl (5).
Two neighboring protein residues (Asn321 and Asn323) are donating
hydrogen bonds to the ND1 of His447 precluding its tautomerization
upon substrate binding to the other neutral form of imidazole.
Additional atomic resolution structural studies on the enzyme
over a broad pH range showed that the NE2 atom of His447 remains
protonated up to at least pH 7.5 (16). This hydrogen bond network
suggests that the role of His447 is as a hydrogen bond donor to
the hydroxyl oxygen atom of the substrate and that it serves to
position the substrate with respect to the flavin. In this arrangement,
the hydrogen atom of the steroid hydroxyl group is directed towards
the side chain of Glu361 and the lone pair of electrons points
towards the protonated NE2 of His447 (Scheme 1). This anti-periplanar
configuration supports a concerted trans-elimination reaction
of proton and hydride. Thus, the substrate O-H bonding electrons
can delocalize into the antibonding orbital of the C-H group to
promote hydride transfer. It is not clear that complete proton
transfer from the hydroxyl group to Glu361 occurs, since the following
isomerization reaction would be enhanced by general acid catalysis
at the oxygen. Moreover, mutation of Glu361 to glutamine in combination
with mutation H447Q only reduced kcat and kcat/Km 3–fold
relative to the single point mutant H447Q (17).
Figure 2: View of the electron
density around H447, pdbentry 1MXT. Green contours represent the
Fo-Fc map contoured at 1.5s and magenta
represents the SigmaA map contoured at 4s.
Uniquely amongst GMC family members, cholesterol oxidase catalyzes
a second non-redox reaction in the active site, isomerization
of a b,g–unsaturated
ketone to an a,b-unsaturated
ketone. This isomerization is a base-catalyzed process as demonstrated
by deuterium transfer studies and mutagenesis (4,18,19). The 4b-hydrogen
is transferred to the 6b position on
the steroid (4,18). Glu361 is strategically positioned over the
b-face to catalyze this isomerization
(9,10) Indeed, mutagenesis to a glutamine shut down isomerization
completely. The reason for this bifunctionality may be the instability
of the cholest-5-en-3-one initially formed upon oxidation of cholesterol.
The cholest-5-en-3-one is particularly susceptible to auto-catalyzed
radical peroxidation and forms cholest-4-en-6-hydroperoxy-3-one
that disproportionates to cholest-4-en-3,6-dione and cholest-4-en-6-ol-3-one
(4). The various reports that different sources of cholesterol
oxidase produce more or less cholest-4-en-6-ol-3-one (20,21) probably
reflect the varying degrees to which different cholesterol oxidases
release cholest-5-en-3-one before isomerization occurs. For example,
in the case of wild–type Streptomyces cholesterol oxidase,
we observed by HPLC that 2% of the product initially formed is
cholest-5-en-3-one which is subsequently degraded to the hydroperoxide
(4).
