Báo cáo khoa học: Cholesterol oxidase: biochemistry and structural features ppt

MINIREVIEW Cholesterol oxidase: biochemistry and structural features Alice Vrielink 1 and Sandro Ghisla 2,3 1 School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, Australia 2 Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy 3 Fachbereich Biologie, University of Konstanz, Konstanz, Germany Introduction Cholesterol oxidases (ChOxs) are bacterial flavopro- teins that catalyze the first step in the degradation of cholesterol. They contain a single molecule of FAD as the redox cofactor and, in one case, possibly FMN. ChOx has emerged as a useful biotechnological tool employed, for example, for the determination of serum cholesterol levels (see accompanying reviews [1,2]). It possesses larvicidal and insecticidal activity [3,4] and might be a factor important for infection with the pathogenic bacterium Rhodococcus equi [5,6]. The enzymatic activity is unique to bacteria and thus ChOx constitutes a potential target for new anti- biotics. The 3D structures of two types of ChOx that show completely different tertiary topologies while catalyzing the same reaction have been solved by crystallography [7,8] and will be discussed in detail below. Depending on the origin, ChOxs differ in their kinetic and redox properties. In addition, the kinetic features of ChOx are tailored to make use of membrane-bound choles- terol as a substrate (see accompanying review [1]). Recently, ChOx has also gained mechanistic interest as Keywords cholesterol oxidase; enzyme kinetics; enzyme mechanism; flavoenzyme; oxygen channel; protein structure; redox catalysis Correspondence A. Vrielink, School of Biomedical Biomolecular and Chemical Sciences, University of Western Australia, 35 Stirling Highway - Crawley, WA, 6009 Australia Fax: +61 8 6488 1148 Tel: +61 8 6488 3162 E-mail: alice.vrielink@uwa.edu.au (Received 27 July 2009, revised 7 September 2009, accepted 14 September 2009) doi:10.1111/j.1742-4658.2009.07377.x Cholesterol oxidases are bifunctional flavoenzymes that catalyze the oxida- tion of steroid substrates which have a hydroxyl group at the 3b position of the steroid ring system. The enzyme is found, in a wide range of bacte- rial species, in two forms: one with the FAD cofactor bound noncovalently to the enzyme; and one with the cofactor linked covalently to the protein. Here we discuss, compare and contrast the salient biochemical properties of the two forms of the enzyme. Specifically, the structural features are dis- cussed that affect the redox potentials of the flavin cofactor, the chemical mechanism of substrate dehydrogenation by active-center amino acid resi- dues, the kinetic parameters of both types of enzymes and the reactivity of reduced enzymes with molecular dioxygen. The presence of a molecular tunnel that is proposed to serve in the access of dioxygen to the active site and mechanisms of its control by a ‘gate’ formed by amino acid residues are highlighted. Abbreviations BsChOx, cholesterol oxidase from Brevibacterium sterolicum containing the FAD cofactor covalently linked to the enzyme; ChOx, cholesterol oxidase; H 2 O 2 , hydrogen peroxide; ReChOx, cholesterol oxidase from Rhodococcus equi, originally identified as a Brevibacterium sterolicum enzyme, containing the FAD cofactor noncovalently bound to the enzyme; SChOx, cholesterol oxidase from Streptomyces SA-COO containing the FAD cofactor noncovalently bound to the enzyme; ShChOx, cholesterol oxidase from Streptomyces hygroscopicus containing the FAD cofactor noncovalently bound to the enzyme. 6826 FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS it was the first flavoprotein for which a molecular tun- nel has been uncovered that has been proposed to serve in oxygen access for the oxidative half-reaction of the enzyme [7,9–11]. Catalysis by cholesterol oxidase ChOx catalyzes three chemical conversions (see Scheme 1 for an overview of the enzymatic steps). The first catalytic conversion, called the reductive half-reac- tion, is the dehydrogenation of the alcohol function at the 3-position of the steroid ring system. The resulting two redox equivalents are transferred to the (oxidized) flavin cofactor that becomes reduced in the process. In the second catalytic step, the reduced flavin reacts with dioxygen to regenerate the oxidized enzyme and hydro- gen peroxide (H 2 O 2 ) (oxidative half-reaction). Finally, the oxidized steroid undergoes an isomerization of the double bond in the steroid ring system, from D5-6 to D4-5, to form the final product cholest-4-en-3-one. In general, this isomerization reaction occurs faster than the release of the intermediate, cholest-5-en-3-one, from the enzyme. The specificity of ChOx for various substrates derived from the cholestane skeleton has been the object of several studies, in particular those describing newly discovered ChOxs (see accompanying review 3 [1]). While the dehydrogenation of the CH–OH func- tion at position 3 of the cholestane is retained, the introduction of functional groups that alter the polar- ity ⁄ hydrophobicity of the same have deleterious conse- quences [12]. By contrast, it is noteworthy that most low-molecu- lar-mass alcohols (e.g. propan-2-ol) are substrates which have the ability to reduce the enzyme within 1 h at a concentration of  1 m [12]. This highlights the concept that ChOx is an alcohol oxidase adapted to accommodate the bulky cholestane frame. Forms of cholesterol oxidase While the presence of the isoalloxazine (flavin) moiety as the redox catalyst at the active center is a common feature amongst ChOxs, three different forms of the cofactor have been identified to date. Figure 1A,B compare sequence alignments, performed using clustalw2 [13], for different types of ChOxs. In the majority of cases FAD is tightly, but noncovalently, bound to the protein (Fig. 1A, reporting the sequence of ChOx from R. equi [17] previously classified as non- covalent ChOx from Brevibacterium sterolicum), while in those cases where the isoalloxazine is covalently attached to the protein, this occurs via a bond linking the 8-methyl group of the isoalloxazine moiety to the polypeptide chain (Fig. 1B). In the case of the enzyme from Brevibacterium, the covalent attachment has been identified through structural and mutagenesis studies to involve the imidazole ND1 atom of a histidine resi- due (His121) [7,14]. In one specific case, the flavin has been reported to be FMN, although the evidence for this does not appear to be conclusive [15]. ChOx proteins belonging to the subfamilies contain- ing either covalent [cholesterol oxidase from B. steroli- cum containing the FAD cofactor covalently linked to the enzyme (BsChOx)] or noncovalent [cholesterol oxidase from Rhodococcus equi, originally identified as a Brevibacterium sterolicum enzyme, containing the FAD cofactor noncovalently bound to the enzyme (ReChOx), cholesterol oxidase from Streptomyces SA-COO containing the FAD cofactor noncovalently bound to the enzyme (SChOx) and cholesterol oxidase from Streptomyces hygroscopicus containing the FAD cofactor noncovalently bound to the enzyme (ShChOx)] forms of FAD have been extensively char- acterized with respect to their biochemical and kinetic properties [12,16]. These exhibit differences in amino acid sequences (Fig. 1), structures [7,8] and redox properties [16]. The structural differences will be out- lined below. In the noncovalent form of the enzyme, release of the cofactor is possible, for example under denaturation conditions such as heating at 90 °C [17]. This indicates the high degree of stability of the FAD–protein complex, largely because of the extensive Scheme 1. Reaction steps catalyzed by ChOx and structures of species involved. The terms ‘Reductive’ and ‘Oxidative’ half-reac- tion refer to the changes of the redox state of the bound flavin coenzyme. Cholest-5-en-3-one does not occur in free form under normal catalytic conditions. Its conversion to the final product, cholest-4-en-3-one, is faster than its formation. A. Vrielink and S. Ghisla Biochemistry of cholesterol oxidase FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS 6827 A Fig. 1. Sequence alignments of different forms of cholesterol oxidase. The sequences were aligned using CLUSTALW2 at http://www. expasy.ch. Sequences were obtained using the protein search algorithm at The National Centre for Biotechnology Information (NCBI). Panel (A) corresponds to sequences for ChOx containing the FAD cofactor noncovalently bound to the enzyme. These include Streptomyces sp. SA-COO (SChOx, S. sp, AAA26719), Rhodococcus equi (R. equ, CAC44897), Vibrio harveyi HY01 (V. har, ZP_01986092), Salinispora arenicola CNS-205 (S. are, YP_001537636), Nostoc punctiforme PCC 73102 (N. pun), Frankia sp. EAN1pec (F. sp, YP_001508197), Mycobacte- rium tuberculosis H37Rv (M. tub, CAB01014), Mycobacterium leprae (M. lep, CAC29897), Corynebacterium urealyticum DSM7109 (C. ure, YP_001800712) and Streptomyces coelicolor (S. coe, NP_628939). Panel (B) corresponds to sequences for ChOx containing the FAD cofac- tor covalently linked to the enzyme. These include Brevibacterium sterolicum (BsChOx, B. ster), Burkholderia cepacia (B. cepa, BAB63263), Burkholderia thailandensis E264 (B. thai, YP_441176), Chromobacterium sp. DS-1 (Chrom, BAG70948) and Rhodococcus erythropolis (R. eryt, ABW74861). Yellow shading indicates regions of the chain that are important in interactions with the FAD cofactor; * are residues that make actual hydrogen bonding contact to the cofactor; and red shading indicates residues that are implicated as playing a role in cataly- sis. The secondary structure elements for a) SChOx and b) BsChOx have been included in red (alpha helices) and blue (beta strands). Biochemistry of cholesterol oxidase A. Vrielink and S. Ghisla 6828 FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS noncovalent interactions between the cofactor and the protein. Apoenzyme of cholesterol oxidase In general ChOx binds the cofactor very tightly, and its (reversible) removal requires rather harsh condi- tions. Wels [16] has studied systematically the prepa- ration of the apoprotein form of ShChOx with the result that most common methods (see Husain & Massey [18] for a review) lead either to incomplete removal of FAD, or to extensive denaturation. Removal of the cofactor in a reversible manner is achieved at very low pH (< 2.0) and the differences in the absorption spectra between free FAD and FAD bound to protein have been monitored spectro- scopically (see Fig. 2). The resulting apoprotein (80% yield) was highly unstable however, and must be reconstituted at high pH (> 8.0) using a large excess of FAD [16]. The apoprotein of a BsChOx mutant, where the histidine residue that forms the covalent link to the flavin moiety has been mutated to an alanine, was obtained by using a high potassium bromide concen- tration: its characterization reveals lower stability compared with the holoenzyme form, suggesting a link between protein stability and covalent linkage between the flavin cofactor and the protein [19,20]. Redox properties While the catalytic dehydrogenation reaction of ChOx in the presence of steroid substrate proceeds via a sin- gle two-electron step from the oxidized flavin form B Fig. 1. (Continued). A. Vrielink and S. Ghisla Biochemistry of cholesterol oxidase FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS 6829 directly to the fully reduced form (see Fig. 2), reduc- tion with artificial electron donors generates first the flavin semiquinone species that is stabilized kinetically in its (red) anionic form [16] (results not shown). The redox properties have been studied for both the ‘cova- lent’ [16,21] and ‘noncovalent’ [22] forms of the enzyme. The midpoint reduction potential, E m , of the covalent form of the enzyme was measured according to the method of Massey [23] and exhibits an E m of )101 mV. Interestingly, in the H121A mutant, in which the cova- lent linkage between the flavin and the peptide chain is absent, the E m is lowered to )204 mV [14]. The 3D structure of the mutant shows that the absence of the covalent link leads to a more planar configuration of the three-membered flavin system, resulting in a more tetrahedral-like geometry for N5 [18]. This difference was proposed to contribute to the observed alteration in reduction potential. The E m for ShChOx has been reported to be )217 mV [16]. Interestingly, SChOx exhibits an E m dependence on pH (E m = )131 mV at pH 7.0 and E m = )73 mV at pH 5.1) [22]. Spectral properties of selected cholesterol oxidases The absorption spectra of flavoproteins are very useful parameters for assessing a variety of properties [24–27]. They reflect the electronic state of the isoallox- azine chromophore and consequently give information about its redox and ionization states. In addition, the spectra are very sensitive to the microenvironment at the active site, such as hydrophobicity ⁄ polarity and the presence of charges ⁄ dipoles. The spectra have thus been used widely to deduce properties of the enzyme [28]. Figure 2 depicts the absorption spectra of ChOx from two different origins in the corresponding oxi- dized and fully reduced states, as well as the spectrum of free FAD. It should be noted that the band in the visible region of the spectrum can vary in its intensity by up to a factor of two, while the position of the maximum may differ by  20 nm. Taken prima facie, the spectrum of oxidized ShChOx would indicate an apolar environment, while that of BsChOx suggests conditions similar to those of free FAD in water. The differences between the fully reduced forms are quite extensive. Thus, the three bands that are posi- tioned near 300, 340 and 400 nm in free reduced flavin [29] appear red-shifted in reduced BsChOx, while an opposite effect underlies the spectra of reduced ShChOx (Fig. 2). By comparison with the spectral properties reported earlier [16], it can thus be deduced that, in the case of ShChOx, the reduced flavin is (mainly) in its anionic form, while in BsChOx either the neutral form or a mixture of both is present. The semiquinone forms similarly have characteristic spectra with maxima in the near-UV range at 372 (ShChOx) and 382 (BsChOx) nm, and 445 (ShChOx) and 485 (BsChOx) nm, which reflect the presence of the chromophore exclusively in its anionic form (results not shown; note that the neutral flavin radical is blue with maxima in the 560–620 nm region, while with both ChOx enzymes no absorption is observable in this area) [16]. Both ShChOx and BsChOx exhibit a weak fluores- cence emission that is maximal at  525 nm and approximately 0.5% that of free oxidized FAD [16]. Interestingly, the emission intensity of the reduced forms of BsChOx and ShChOx is  fourfold higher and shifted towards 490 nm [16]. Structures Overall structure As pointed out above, ChOx has been identified in a number of bacteria and these flavoenzymes exhibit lar- gely differing sequences (Fig. 1A,B) that suggest large structural differences between the proteins. For many FAD-containing enzymes, a consensus sequence of repeating glycine residues (GXGXXG) followed by a 300 350 400 450 500 550 600 FAD (ox) BCO (ox) BCO (red) SCO (ox) SCO (red) FAD (red) Wavelength (nm) 448 Extinction coefficient (M –1 ·cm –1 ) 12 × 10 3 4 × 10 3 8 × 10 3 388 467 362 350 Fig. 2. Absorption spectra of ChOx from Streptomyces hygroscopi- cus (blue) and Brevibacterium sterolicum (red) in their oxidized and fully reduced states. Enzyme solutions were  10 l M in 0.1 M potas- sium phosphate (pH 7.5) and at 25 °C. The fully reduced forms of ShChOx and BsChOx were obtained upon the addition, anaerobically, of small amount of excess cholesterol. The spectra of FAD in its oxidized [87] and fully reduced forms are reported for comparison. Biochemistry of cholesterol oxidase A. Vrielink and S. Ghisla 6830 FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS D ⁄ E approximately 20 residues further along the pri- mary sequence, is characteristic of a nucleotide-binding fold (in other words, a motif that facilitates interac- tions between the bound nucleotide and the protein) [30,31]. The consensus sequence of glycine residues allows binding of the charged diphosphate moiety of the nucleotide near to the N-terminal end of a helix, with stabilization exerted through the helix dipole effect [32]. The presence of an aspartate or glutamate side chain  20 residues further along the protein chain enables hydrogen bond interaction between the 2¢ and 3¢ hydroxyl groups of the nucleotide ribose moi- ety and the carboxylate group of the side chain. These features are characteristic of many nucleotide-binding proteins and appear to be critical in forming the neces- sary interactions between the nucleotide and the pro- tein to favor correct binding of the cofactor. The noncovalent form of ChOx exhibits an almost identical consensus sequence of glycines (G17-X-G19-X-G21- G ⁄ A22) followed by a glutamate (E40), indicating the presence of a nucleotide-binding fold (see Fig. 1A, the numbering of residues corresponds to the SChOx sequence); however, in the case of the covalently bound ChOx this consensus is notably absent (Fig. 1B), suggesting the likely absence of a nucleotide- binding fold for this form of the enzyme. Nevertheless, the FAD cofactor is bound tightly, even when the covalent link with the apoprotein moiety is removed (see above). Indeed, as noted above, the sequence iden- tity can be used as a predictive measure of the mode of flavin binding to the enzyme: covalent versus non- covalent linkage. High-resolution crystal structures were determined for two noncovalent forms of the enzyme (SChOx and ReChOx) [8,33] and for one of the covalent forms (BsChOx) [7], providing a view of the overall folds for each protein (Fig. 3). The overall topologies for each form of the enzyme vary significantly from each other (Fig. 3). Although both enzymes have been defined as being composed of two domains based on function (FAD-binding domain and sub- strate-binding domain), they can also be considered as single-domain proteins based on topology because the protein chain meanders back and forth between the regions that provide the binding features for the cofactor and the binding features for the substrate as well as the catalytic residues. The noncovalent ChOx belongs to the glucose-methanol-choline (GMC) oxi- doreductase flavoenzyme family [34] whereas the covalent enzyme belongs to the vanillyl-alcohol oxi- dase (VAO) family [35]. The structure of noncovalent ChOx shows the cofac- tor binding motifs, containing the consensus sequence of glycines (as discussed above) as well as two further regions of conserved glycines (Figs 1A and 4A), all of which allow a close approach of the protein main A B Fig. 3. 3D structure of ChOx. An overall view of the secondary structure elements for (A) SChOx and (B) BsChOx is shown. The FAD cofactor is presented in ball-and-stick representation. A. Vrielink and S. Ghisla Biochemistry of cholesterol oxidase FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS 6831 chain to the phosphate oxygen atoms of the cofactor, thus facilitating hydrogen bond interactions. The noncovalent enzyme possesses the characteristic nucleotide-binding fold (Rossmann fold) consisting of a b-pleated sheet sandwiched between a-helices and containing the motif needed for binding the cofactor (Fig. 3A). The diphosphate group of the cofactor is positioned closely packed to the N terminus of the first a-helix of the protein (Fig. 4A) where the conserved GXGXG glycine residues are located. Side chains larger than glycines at this critical loop between the N-terminal end of the helix and the first b-sheet would result in positioning the flavin diphosphate moiety further away from the chain and hence outside the polarization field resulting from the helix dipole. Hence, the glycine residues are strategically positioned to allow the helix dipole to stabilize the negative charge of the cofactor. In the covalent form of the enzyme, the diphosphate moiety is localized in a pocket made by the residues found between the third and fourth b-strands of a four-stranded b-pleated sheet (Fig. 4B). Interaction between the phosphate oxygens and the protein involve main chain nitrogen atoms of residues in this loop region. Furthermore, this loop contains His121, the side chain of which covalently connects to the cofac- tor. In contrast to the noncovalent form of the enzyme, the covalently bound form does not exhibit hydrogen bond interactions between the ribose hydro- xyl groups of the cofactor and the protein. The ribityl chain of the cofactor also makes much fewer hydrogen bond interactions with the protein in the covalently bound form compared with the noncovalently linked form. Covalent linkages of FAD to proteins have shown that, in addition to histidine residues, cysteine, tyrosine and threonine side chains can be involved (see Heuts et al. [36] for a review of covalent linkages between flavoproteins and their cofactors). A compari- son of the sequences for other covalently bound forms of the enzyme (Fig. 1B) predicts that, in all cases, the linkage between the cofactor and the protein is to a histidine side chain. Vanillyl-alcohol oxidase, another example of a flavoenzyme containing covalently bound flavin, also utilizes a histidine for the covalent attach- ment; however, in this case the linkage involves the NE2 atom of the histidine residue [35]. The structure A B C D E F Fig. 4. Structural views of different forms of ChOx. Interactions between the protein and the FAD cofactor are shown for (A) the noncova- lently linked ChOx (SChOx) and (B) the covalently linked enzyme (BsChOx). Hydrogen bonds between the cofactor and the protein are shown as dashed lines. Models of the bound steroid in the active sites of (C) SChOx and (D) BsChOx. Active site residues of (E) SChOx and (F) BsChOx. Biochemistry of cholesterol oxidase A. Vrielink and S. Ghisla 6832 FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS of BsChOx reveals that the covalent linkage is made to the ND1 atom of the histidine side chain. However, this bond is not a prerequisite for flavin binding because in the H121A mutant, FAD is bound tightly and forms a catalytically competent holoprotein in which the redox potential of the cofactor is lowered by  100 mV (see above) [14,21]. The structure reveals changes in the conformation of the isoalloxazine ring system that are proposed to affect the redox chemistry [14]. In addition to the differences in the cofactor bind- ing motifs between the covalent and noncovalent ChO- xs there are also large differences in the overall topology. These differences are evident through the sequence diversity seen between the two enzyme forms (Fig. 1A,B). The active site Despite the topological differences in the two enzyme forms, both contain a large buried hydrophobic pocket that is able to accommodate the steroid ring system (Fig. 4C ⁄ D). In both forms of enzyme the binding site for the steroid is sealed off from the external environ- ment of the protein by a number of loops, which exhi- bit higher mobility than the rest of the protein structure. This suggests that these loops must re-orient to allow the steroid substrate to enter the hydrophobic pocket. Comparisons of the structures of SChOx and ReChOx show differences in the nature of the loops; in SChOx the loops are more rigid in nature as well as containing a amphipathic helical turn, whereas in ReChOx the loops are more extended, lack secondary structure elements and exhibit higher temperature fac- tors [33]. The higher mobility of the substrate entry loops in ReChOx compared with SChOx correlates with the observed elevated K m values for both choles- terol (K m =3lm for SChOx and > 100 lm for ReChOx) and dehydroisoandosterone (K m = 27.5 lm for ChOx and 400 lm for ReChOx) in ReChOx [33]. Indeed, the increased rigidity of the loops in SChOx pre-orients the residues needed for binding the 8-car- bon isoprenoid tail at C17 of the substrate and thus increases the efficiency of the enzyme for catalysis. The isoalloxazine ring system is located at the ‘base’ of the pocket. The active site is on the re face of the isoalloxazine for the noncovalent form of the enzyme and on the si face for the covalent enzyme form. ChOx requires a number of features for efficient catalysis. First, the steroid must be correctly oriented relative to the cofactor for hydride transfer from the steroid C3 site to the isoalloxazine N5 site. Second, side chain functional groups for two catalytic steps are needed: (a) a base to accept the steroid C3-O-H proton during oxidation and (b) a base for proton transfer during the isomerization reaction. Finally, conditions must be met for binding and conversion of molecular oxygen to peroxide during the oxidative half-reaction. Despite these common requirements, the active sites of the two forms of ChOx are highly divergent. Indeed, the differences in sequences and structures for both enzymes are also evident in terms of residues identified as playing roles in catalysis. Chemical mechanisms Dehydrogenation From a chemical point of view the reductive half-reac- tion catalyzed by ChOx involves the dehydrogenation of a nonactivated alcohol to generate the correspond- ing carbonyl function, with the resulting redox equiva- lents transferred to the bound flavin cofactor, as shown in Scheme 2. This type of reaction is catalyzed by several classes of flavoproteins, such as the methanol (or alcohol) oxidases [37,38] (including e.g. alditol oxidase [39]), those acting on a-OH-carboxylic acid oxidases (e.g. lactate oxidases ⁄ dehydrogenases [40]) and those that dehydrogenate the hydroxyl group of cyclic carbohy- drates (e.g. glucose oxidase [41]). While this type of reaction is common in biochemi- cal systems, its catalytic mechanism has been much debated [42]. In the reaction, the key step is the rup- ture of the kinetically stable C-H bond for which three modes are conceivable [43]: (a) a homolytic fission (radical mechanism), (b) a heterolytic mechanism, in which the hydrogen is abstracted as H + (carbanion mechanism), and (c) a heterolytic mechanism, in which expulsion of a hydride generates (transiently) a carbe- nium ion (hydride mechanism). These basic modes have been discussed in detail elsewhere [44]. While ear- lier studies tended to favor mechanism (a) [37,38] most authors now agree on mechanism (c), the so-called ‘hydride transfer’ for this type of reaction [9,33,45–47]. This mechanism is very common in biochemical sys- tems and is operative also for the dehydrogenation of the same CH–OH functionality by pyridine nucleotide [48,49] and quinoprotein-dependent enzymes [50]. Two prerequisites are necessary for dehydrogenation via this mechanism: correct alignment of the orbitals involved; Scheme 2. Overview of the reductive half-reaction catalyzed by ChOx. A. Vrielink and S. Ghisla Biochemistry of cholesterol oxidase FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS 6833 and a base that accepts the –OH proton (as shown in Scheme 3). One reason for the requirement of a base in the dehydrogenation of the CH-OH group [51] is because of the high pK a of the hydroxyl group (> 15) and the fact that the reaction appears to involve a concerted rupture of the O-H and C-H bonds. A discussion of the role of specific amino acid residues in catalysis fol- lows below. In contrast to the dehydrogenation of the CH-OH group, for oxidation of the CH-NH 2 function- ality (such as in amino acids), the presence of a base is not mandatory [52] because of the lower pK b (8–10) for this group. As argued convincingly by Klinman and coworkers, the transfer of the hydride to the acceptor involves a high degree of tunneling, the extent of which might depend on the specific enzyme [48,53]. In the case of SChOx, the structure of a steroid- bound complex (Fig. 4C) has provided initial insights into the specific roles of catalytic residues and the mechanisms of both oxidation and isomerization [54]. Mutagenesis and kinetic analyses, as well as further structural studies, have helped to identify the role in catalysis of a number of key residues. These studies have suggested that His447 plays a role in substrate orientation and the correct orbital alignment for de- protonation of the C3-OH proton and hydride transfer to the cofactor [9,33,55,56]. Recent structural studies have suggested that Tyr446 may act by exerting steric pressure on the isoalloxazine ring moiety, thereby affecting the ability of the cofactor to become reduced [57]. This re-orientation of the tyrosine side chain, and the resulting distortion of the flavin ring system, is induced by substrate binding at the active site and is an example of an induced-fit mechanism for the modu- lation of the redox potentials of the cofactor. Asn485 acts to stabilize the reduced cofactor and, through a movement towards the reduced isoalloxazine ring system, also allows access of oxygen to the active site via the oxygen channel (see below) for the oxidative half-reaction [58]. A definitive identification of the base needed for dehydrogenation is still somewhat unclear. A likely candidate is Glu361 [9]. However, this residue has also been shown to be required as the base for isomeriza- tion chemistry [59,60] (see below). Invoking Glu361 as the base for both catalytic reactions requires the pro- ton abstracted from the C3-OH group to be trans- ferred elsewhere after dehydrogenation chemistry has occurred so that the glutamate can act as the base for the subsequent isomerization step. The mechanism and kinetics of this proton transfer step are not yet clearly understood. It may be that it is transferred directly to an incoming oxygen molecule as part of the oxidative half-reaction. This is possible because the glutamate side chain is positioned near to the point where the oxygen channel enters the active site. Alternatively, the proton may be transferred to C(4)=O of the isoalloxa- zine moiety of the cofactor before reacting with dioxy- gen; however, this would require an intermediate residue to shuttle the proton the 5 A ˚ distance from the glutamate carboxylate to the isoalloxazine ring. The covalent ChOx has not been crystallized in the presence of a steroid substrate or a substrate analogue. The binding site for the substrate is much more hydro- philic in nature than the noncovalent enzyme because of the presence of a large number of charged side chains (Fig. 4F). Two charged residues (Glu475 and Arg477) are found to adopt two distinct conforma- tions. The positions of their side chains are correlated with one another, suggesting that the movements they make are concerted. Based on the structure and on a model of the steroid substrate bound to the enzyme, it has been inferred that Glu475 is the base for proton abstraction of the steroid C3-OH in the reductive half- reaction of BsChOx [7]. Isomerization The isomerization step of the cholest-5-en-3-one is the second reaction catalyzed by most ChOx proteins in which the hydrogen at position C(4) is transferred to position C(6) [59]. The theoretical requirements for the isomerization step of catalysis are represented in Scheme 4. The C-H group at position 4 of the cholestane ring is activated (acidified) by the flanking carbonyl group. This facilitates abstraction of the hydrogen as H + by an appropriately positioned base to yield a transient enolic species. Reprotonation at position 6 then leads to the final cholest-4-en-3-one product. The isomerization step of the intermediate cholest-5- en-3-one to final product is slow (0.3 s )1 ) in the case of reduced ShChOx; however, the rate of this conver- sion is approximately three orders of magnitude faster with the oxidized form [12]. From this it can be concluded that with ShChOx in the catalytic mecha- nism depicted in Scheme 4, the isomerization follows Scheme 3. Depiction of the requirements for efficient dehydroge- nation in ChOx (‘Acc’ stands for the electron acceptor FAD). Biochemistry of cholesterol oxidase A. Vrielink and S. Ghisla 6834 FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS re-oxidation and is not rate limiting for catalysis [12,16]. With BsChOx, the isomerization proceeds at approximately the same rate ( 20–30 s )1 ) with the oxidized or reduced enzyme forms [14] and is close to the rate of flavin reduction at saturating substrate concentration [16]. This reaction has been studied in detail for SChOx by the group of Sampson [55,59,60]: H + abstraction occurs stereospecifically on the b-face of the steroid ring system, is mediated by Glu361 and the label is retained in the product. In an identical manner to SChOx, a glutamate is thought to play the role of a base in both the oxida- tion and in the proton-transfer mechanism of isomeri- zation in BsChOx. The model of the enzyme with substrate bound reveals the side chain of Glu475 on the b-face of the steroid substrate, well positioned to abstract the C4 proton and reprotonate at C6 of the enolic intermediate [7]. It has been proposed that Glu311 and Glu475 are involved in a proton-transfer mechanism to allow the proton removed from the ste- roid C3-OH during the reductive half-reaction to be positioned such that it can react with molecular oxy- gen during the oxidative half-reaction [7]. This proton- transfer mechanism enables Glu475 to take on the role of a base for isomerization [10]. Kinetics General aspects It should be noted that the several publications describing the isolation of ChOx from various sources in general give some rudimentary indications about the activity of the purified protein. Because the catalytic activity of ChOx is highly dependent on the buffer composition, pH and on the type and concentration of surfactant used to solubilize the substrate (which in turn affects micelle composition and dynamics), a com- parison and discussion of such parameters is problem- atic (see also accompanying review [1]). Pollegioni et al. [61] have addressed these topics in some detail and have also studied the stability of the ShChOx and BsChOx in the presence of different concentrations of some organic solvents. In essence, they conclude that every single one of the named parameters will affect the catalytic activity to some extent. Nishiya et al. studied the effect of specific detergents on a mutant of a Streptomyces ChOx and detected a change of mecha- nism compared with wild-type ChOx [62]. Ahn and Sampson [63] studied the effect of lipid structure on the activity of ChOx and concluded that ‘enzymatic activity directly reflects the facility with which choles- terol can move out of the membrane’. A study of ChOx activity as a function of assay type, structure of the steroid substrate and presence of solubilizers was conducted by Gadda et al. [16], and the deduced parameters are listed in Table 1. A more detailed study by the same groups compared the kinetic mechanisms of ShChOx and BsChOx [12]. Therein it was concluded that BsChOx acts via a ping- pong mechanism, whereas the catalytic pathway of ShChOx is sequential. Xiang et al. [64] have generated models of the expected Michaelis complex of ChOx and cholesterol and used this information to design mutants expected to exhibit activities different from those of the parent enzyme. The results were inconclusive in this respect, although they did indicate that the loss of active-site water was the predominant source of binding energy. Sampson et al. [65] have also studied the role of a spe- cific active-site loop of ChOx. The activity of the mutant with cholesterol in a vesicle is decreased 2800- fold compared with wild-type enzyme, whereas isomer- ization activity is retained. The authors conclude that ‘the loop is important for movement of cholesterol from the lipid bilayer’. In an analogous study, Toyama and coworkers [66] attempted to compare the activities of the ChOxs by implementing mutations that were expected to yield convergent active sites. Some of the mutants indeed lead to significantly altered catalytic parameters for various substrates and consequently to a different spec- ificity spectrum. The reductive half-reaction The reductive half-reaction was investigated in com- parative manner for ShChOx and BsChOx using the stopped-flow technique [12]. Both enzymes are reduced *H O O O ~B| *H ~B*H + ~B| 4 6 6 4 Scheme 4. General mechanism for the isomerization reaction of ChOx: B| is a base that abstracts and redonates a H + to the intermediate. Only the reactive portion of the steroid substrate is shown. A. Vrielink and S. Ghisla Biochemistry of cholesterol oxidase FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS 6835 [...]... Purcell JP (2001) Expression and chloroplast targeting of cholesterol oxidase in transgenic tobacco plants Plant Physiol 126, 1116–1128 5 Machang’u RS & Prescott JF (1991) Purification and properties of cholesterol oxidase and choline phosphohydrolase from Rhodococcus equi Can J Vet Res 55, 332–340 6 Fuhrmann H, Dobeleit G, Bellair S & Guck T (2002) Cholesterol oxidase and resistance of Rhodococcus... 239, 1–12 29 Ghisla S, Massey V, Lhoste JM & Mayhew SG (1974) Fluorescence and optical characteristics of reduced flavins and flavoproteins Biochemistry 13, 589–597 30 Eventoff W & Rossmann MG (1975) The evolution of dehydrogenases and kinases CRC Crit Rev Biochem 3, 111–140 31 Ohlsson I, Nordstrom B & Branden CI (1974) Structural and functional similarities within the coenzyme binding domains of dehydrogenases.. .Biochemistry of cholesterol oxidase A Vrielink and S Ghisla Table 1 Kinetic parameters for turnover by BsChOx and ShChOx using different substrates and different assays The data were estimated for 25 °C and pH 7.5 as described previously [61] Three different assays were used that rely on the spectrophotometric... Chen L, Lyubimov AY, Brammer L, Vrielink A & Sampson NS (2008) The binding and release of oxygen and hydrogen peroxide are directed by a hydrophobic tunnel in cholesterol oxidase Biochemistry 47, 5368– 5377 12 Pollegioni L, Wels G, Pilone MS & Ghisla S (1999) Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum Eur J Biochem 264, 140–151 6840 13 Larkin... IM, Wilm A, Lopez R et al (2007) Clustal W and Clustal X version 2.0 Bioinformatics 23, 2947–2948 14 Lim L, Molla G, Guinn N, Ghisla S, Pollegioni L & Vrielink A (2006) Structural and kinetic analyses of the H121A mutant of cholesterol oxidase Biochem J 400, 13–22 15 Isobe K, Shoji K, Nakanishi Y, Yokoe M & Wakao N (2003) Purification and some properties of cholesterol oxidase stable in detergents from... (2009) Cholesterol oxidase: physiological functions FEBS Journal 23, doi:10.1111/ j.1742-4658.2009.07378.x 2 Pollegioni L, Piubelli L & Molla G (2009) Cholesterol oxidase: biotechnological applications FEBS Journal 23, doi:10.1111/j.1742-4658.2009.07379.x 3 Purcell JP, Greenplate JT, Jennings MG, Ryerse JS, Pershing JC, Sims SR, Prinsen MJ, Corbin DR, Tran M, Sammons RD et al (1993) Cholesterol oxidase:. .. to ligand binding in cholesterol oxidase Protein Sci 16, 2647–2656 58 Yin Y, Sampson NS, Vrielink A & Lario PI (2001) The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase Biochemistry 40, 13779–13787 59 Kass IJ & Sampson NS (1995) The isomerization catalyzed by Brevibacterium sterolicum cholesterol. .. structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity J Mol Biol 326, 1635–1650 10 Piubelli L, Pedotti M, Molla G, Feindler-Boeckh S, Ghisla S, Pilone MS & Pollegioni L (2008) On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in Brevibacterium sterolicum cholesterol oxidase... conformation and (B) the ‘channel closed’ conformation An oxygen molecule is observed in the ‘channel open’ conformation Unquestionably, biochemical and structural studies on ChOx have played a forerunner role in the understanding of various aspects of flavoenzyme redox catalysis, notably as it relates to flavoprotein oxidases This might apply particularly for the insights into the mechanism for re-oxidation and. .. important role in infection [5,6] and the fact that it is unique to bacterial species, opens up an entirely new field It is to be expected that future studies will focus on ChOx potential as a target for new FEBS Journal 276 (2009) 6826–6843 ª 2009 The Authors Journal compilation ª 2009 FEBS 6839 Biochemistry of cholesterol oxidase A Vrielink and S Ghisla antibiotics and thus on the design of specific . MINIREVIEW Cholesterol oxidase: biochemistry and structural features Alice Vrielink 1 and Sandro Ghisla 2,3 1 School of Biomedical, Biomolecular and Chemical. have been included in red (alpha helices) and blue (beta strands). Biochemistry of cholesterol oxidase A. Vrielink and S. Ghisla 6828 FEBS Journal 276 (2009)