Computational site-directed mutagenesis studies of the role of the hydrophobic triad on substrate binding in cholesterol oxidase (original) (raw)
Related papers
Cholesterol oxidase (ChOx), a member of the glucose-methanol-choline (GMC) family, catalyzes the oxidation of the substrate via a hydride transfer mechanism and concomitant reduction of the FAD cofactor. Unlike other GMC enzymes, the conserved His447 is not the catalytic base that deprotonates the substrate in ChOx. Our QM/MM MD simulations indicate that the Glu361 residue acts as a catalytic base facilitating the hydride transfer from the substrate to the cofactor. We find that two rationally chosen point mutations (His447Gln and His447Asn) cause notable decreases in the catalytic activity. The binding free energy calculations show that the Glu361 and His447 residues are important in substrate binding. We also performed high-level double-hybrid density functional theory simulations using small model systems, which support the QM/MM MD results. Our work provides a basis for unraveling the substrate oxidation mechanism in GMC enzymes in which the conserved histidine does not act as a base. Cholesterol oxidase (ChOx) is a flavoenzyme that catalyzes the oxidation of cholesterol to 5-cholesten-3-one and the subsequent isomerization to form the final 4-cholesten-3-one product. The enzyme from Streptomyces is a member of the glucose-methanol-choline (GMC) family and contains a single flavin adenine dinucleotide (FAD) cofactor that is non-covalently bound to the protein. Apart from its physiological functions in bacterial metabolism, pathogenesis, and macrolide biosynthesis 1 , ChOx has also been employed as a useful biotechnological tool, for example, for the determination of serum cholesterol levels 2,3. A number of experimental studies investigated the structure and catalytic mechanism of ChOx over the past two decades 4-11. The active site of ChOx is shown in Fig. 1a. With regard to the enzymatic mechanism, Sampson et al. 7-11 identified three residues (Glu361, His447, and Asn485) that are significant for the enzymatic activity and Vrielink et al. 4,5 revealed that Gly120 also plays an important role in the catalytic mechanism. Mutations of the Glu361 residue in the active site to glutamine and aspartate result in 31-fold 9 and 14-fold 8 reductions, respectively, in the k cat value for the oxidation of the substrate compared to the wild-type (WT) enzyme. This suggests that the carboxylic acid residue at this position may be important for the oxidative activity 6. The His447 residue is located in the active site and the NE2 hydrogen forms a hydrogen bond with the hydroxyl group of the substrate. Mutations of His447 to glutamine and asparagine result in 140-fold and 4400-fold reductions in the k cat value respectively 9 , relative to the WT enzyme, suggesting that His447 plays a significant role in the catalytic activity. However, the double mutant, His447Gln/Glu361Gln, exhibits a 500-fold decrease in the k cat value relative to the WT enzyme 10 , which was 3-fold slower than that for the His447Gln single-mutant. These mutagenesis results indicate that factors affecting the oxidation rate of the two separate mutants, His447Gln and Glu361Gln, are not entirely additive and may be the result of structural perturbations rather than the absence of an active site base. We note that the double mutation does not completely prevent the hydride transfer implying that possibly the FAD is sufficiently electrophilic to oxidize the substrate without complete proton abstraction 6. In addition, other structural studies have suggested that Asn485 forms an N-H•••π electrostatic interaction
Journal of Biological Chemistry, 2005
Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His 69 residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (ϳ3 versus ϳ5 M, respectively), and the mutant protein had a melting temperature ϳ10-15°C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 M urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.
Archives of Biochemistry and Biophysics, 2002
Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k cat of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k cat of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k cat for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor b (PDGFRb) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRb-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRb-dependent pathway tested. Ó
Protein Science, 2008
The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild-type enzyme, but does not modify the overall structure of the enzyme. We used CO as a model system to dissect the changes due to the elimination of the covalent link between the flavin and the protein (by comparing the wild-type and H69A CO holoproteins) with those due to the elimination of the cofactor (by comparing the holo-and apoprotein forms of H69A CO). The apoprotein of H69A CO lacks the characteristic tertiary structure of the holoprotein and displays larger hydrophobic surfaces; its urea-induced unfolding does not occur by a simple two-state mechanism and is largely nonreversible. Minor alterations in the flavin binding region are evident between the native and the refolded proteins, and are likely responsible for the low refolding yield observed. A model for the equilibrium unfolding of H69A CO that also takes into consideration the effects of cofactor binding and dissociation, and thus may be of general significance in terms of the relationships between cofactor uptake and folding in flavoproteins, is presented.
The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.
Biochemistry, 2008
The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, solved at sub-Ångstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To understand the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or the tunnel residues Phe359 or Gly347 to Trp or Asn, reduces the catalytic efficiency of oxidation. The K mO2 increases from 300 ± 35 μM for the wild-type enzyme to 617 ± 15 μM for the F359W mutant. The k cat for the F359W mutant catalyzed reaction decreases 13-fold relative to the wild-type catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Hydride transfer from the sterol to the flavin prosthetic group is no longer rate limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel-mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively are consistent with the cooperative kinetics. In the atomic resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is only observed in a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.
Cholesterol oxidase: ultrahigh-resolution crystal structure and multipolar atom model-based analysis
Acta Crystallographica Section D Biological Crystallography, 2015
Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr. For further information see http://journals.iucr.org/services/authorrights.html Acta Cryst. (2015). D71, 954-968 Zarychta et al. · Cholesterol oxidase research papers 954 http://dx.
Acta crystallographica. Section D, Biological crystallography, 2014
Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occurs via a hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Å resolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Å resolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density p...
The importance of Glu361 position in the reaction catalyzed by cholesterol oxidase
Bioorganic & Medicinal Chemistry Letters, 1998
Cholesterol oxidase stereospecifically isomerizes cholest-J-en-3-one to cholest-4-cn-3-one. When the base catalyst for isomerization, Glu 36~, is mutated to Asp, the rate of deprotonation of cholcst-5-en-3-one is not affected, but protonation of the dienolic intermediate becomes rate-limiting. This may be a consequence of the large distance between the catalytic base and carbon-6 of the intermediate in the mutant enzyme.