Die Cholesterol Oxidase ist ein Flavoprotein, das beim Steroidmetabolismus eine zentrale Rolle spielt und molekularen Sauerstoff als Elektronenakzeptor verwendet. Das Enzym wird bei der klinischen Diagnostik eingesetzt (Bestimmung von Cholesterin im Serum) und hat Potential für Anwendungen bei der Schädlings-
bekämpfung. Aus der von uns kürzlich ermittelten dreidimensionalen Struktur ist ein schmaler, hydrophober Kanal erkennbar, der das Aktivzentrum mit dem Lösungsmittel verbindet. Im Kanal ist ein Glu-Arg Aminosäurepaar positioniert, das in Abhängigkeit von der Anwesenheit von Liganden und vom Redoxzustand des Kofaktors eine "offene" oder eine "geschlossene" Konformation einnehmen kann.
Daraus wurde die Hypothese abgeleitet, dass der Kanal der Diffusion von molekularem Sauerstoff dient. Hierbei würde das Aminosäurepaar im Sinne eines Tors Bestandteil eines Mechanismus sein, der die Reaktivität des Enzyms mit Sauerstoff moduliert. Kanäle und Domänen mit ähnlicher, postulierten Funktion sind kürzlich bei Hämproteinen identifiziert worden. Es ist somit denkbar, dass diese Art der Regulation des Sauerstoffzugangs einen elementaren Mechanismus widerspiegelt dem allgemeine Bedeutung zukommt. Beim vorliegenden Projekt soll die Cholesterol Oxidase als Modellsystem für die Verifizierung des genannten Postulats eingesetzt werden. Hierbei soll eine Kombination von molekularbiologischen (Mutagenese), und biochemischen Methoden mit der Röntgenstrukturaufklärung von Proteinen zur Anwendung kommen.
Cholesterol oxidase is a flavoprotein involved in steroid metabolism that uses oxygen as electron acceptor. It has widespread use in clinical diagnosis (determination of serum cholesterol) and has a potential as a larvicide and pest control agent. Its recently solved 3D-structure at high resolution has revealed the presence of a hydrophobic channel that connects the active center with solvent, and has properties suitable for the permeation of molecular oxygen. At the connection of this channel to the active site there is a pair Glu-Arg positioned such as to control access through the channel. The pair can assume on "open" or a "closed" conformation depending on the presence of ligands at the active center and the redox state of the cofactor. It is postulated that this set-up is part of a mechanism for the control of oxygen reactivity. Similar channels, postulated to have analogous function, have been detected most recently also in heme proteins. Protein mediated recognition of oxygen and control of access to specific centers might thus be a general principle that is only beginning to emerge. We propose to use the enzyme cholesterol oxidase as a model to verify this basic principle and whether is has general validity. A combination of directed mutagenesis, biochemical techniques, and X-ray crystallography shall be used for the project.
- FB Biologie
|(2001): Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair Journal of Biological Chemistry ; 276 (2001), 32. - S. 30435-30441. - ISSN 0021-9258. - eISSN 1083-351X|
Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair
Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-Å resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu475, located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.
|(2001): Cholesterol oxidase from Brevibacterium sterolicum : the relationship between covalent flavinylation and redox properties Journal of Biological Chemistry ; 276 (2001), 21. - S. 18024-18030. - ISSN 0021-9258. - eISSN 1083-351X|
Cholesterol oxidase from Brevibacterium sterolicum : the relationship between covalent flavinylation and redox properties
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 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 8α-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.
|(1999): Cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum : effect of surfactants and organic solvents on activity Biotechnology and Applied Biochemistry ; 30 (1999). - S. 27-33|
Cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum : effect of surfactants and organic solvents on activity
We have studied systematically the effect of the non-ionic surfactants Thesit and Triton X-100, and of propan-2-ol (used as a substrate solubilizer) on the activity of the cholesterol oxidases from Streptomyces hygroscopicus (SCO) and Brevibacterium sterolicum (BCO). Low concentrations of Thesit lead to an activity increase with both enzymes; at higher surfactant concentrations the opposite effect occurs. Triton X-100 inactivates both enzymes at all concentrations. It is deduced that these surfactants exert their effects by interaction with the enzymes and not by affecting micellar phenomena. The effect of propan-2-ol on SCO, in contrast with that on BCO, depends on the buffer concentration (potassium phosphate). Other organic solvents induce results similar to those obtained with SCO and propan-2-ol. A significant difference between the two cholesterol oxidases emerges when stability is tested at 25 °C and in the presence of different concentrations of propan-2-ol: BCO activity is rapidly inactivated, whereas SCO still has 70% of the initial activity after 5 h in the presence of 30% propan-2-ol. From our results, SCO seems to be the catalyst of choice in comparison with BCO for the exploitation of cholesterol oxidases in biotechnology and applied biochemistry.
|(1997): Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum European Journal of Biochemistry ; 250 (1997), 2. - S. 369-376. - ISSN 0014-2956. - eISSN 1432-1033|
Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum
The FAD-containing enzyme cholesterol oxidase catalyzes the oxidation and isomerization of 3β-hydroxysteroids having a trans double bond at Δ5-Δ6 of the steroid ring backbone to the corresponding Δ4-3-ketosteroid. Two representative enzymes of this family, namely cholesterol oxidase from Streptomyces hygroscopicus (SCO) and the recombinant enzyme from Brevibacterium sterolicum (BCO) expressed in Escherichia coli, have been characterized herein in their chemical, physical, and biochemical properties. In the native form, both enzymes are monomeric (55 kDa), acidic (pI 4.4 5.1) and contain oxidized FAD (peaks in the 370 390-nm and 440 470-nm regions). Marked differences exist between the oxidized, reduced, and (red) anion semiquinone spectra of the two enzymes, suggesting substantial differences in the flavin microenvironment. Both enzymes form reversibly flavin N(5)-sulfite adducts via measurable kon and koff steps. BCO has a higher affinity for sulfite (Kd≈ 0.14 mM) compared to SCO (≈24 mM). This correlates well with the midpoint redox potentials of the bound flavin, which in the case of BCO are about 100 mV more positive than for SCO. Both enzymes show a high pKa (≈11.0) for the N(3) position of FAD. With both enzymes, the rearrangement of 5-cholesten-3-one to 4-cholesten-3-one is not rate limiting indicating that the rate-limiting step of the overall reaction is not the isomerization. The absence of the double bond in the steroid molecule does not significantly affect turnover and affinity for the substrate, whereas both these parameters are affected by a decreasing length of the substrate C17 chain.
|Deutsche Forschungsgemeinschaft||683/03||keine Angabe|
|Laufzeit:||01.11.2003 – 31.12.2006|