Lim Louis, Molla Gianluca, Guinn Nicole, Ghisla Sandro, Pollegioni Loredano, Vrielink Alice
Department of Chemistry and Biochemistry, Sinsheimer Laboratory, University of California at Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064, USA.
Biochem J. 2006 Nov 15;400(1):13-22. doi: 10.1042/BJ20060664.
Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a approximately 100 mV decrease in the midpoint redox potential and a approximately 40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024-18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 A resolution (1 A identical with 0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180 degrees about chi1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.
胆固醇氧化酶是一种单体黄素酶，它催化胆固醇氧化生成胆甾 - 5 - 烯 - 3 - 酮，随后异构化为胆甾 - 4 - 烯 - 3 - 酮。来自短杆菌属的该酶含有与His121共价结合的FAD辅因子。先前已证明，与野生型酶相比，H121A替换导致中点氧化还原电位降低约100 mV，周转数降低约40倍[Motteran、Pilone、Molla、Ghisla和Pollegioni（2001年）《生物化学杂志》276，18024 - 18030]。对H121A突变酶的详细动力学分析表明，周转数的降低主要是由于黄素还原速率常数相应降低，而再氧化反应仅略有改变，异构化反应不受该替换影响且先于产物解离。以1.7 Å分辨率（1 Å等同于0.1 nm）测定的突变蛋白的X射线结构显示，蛋白质的整体折叠仅发生微小变化，即：两个环有轻微移动，一个色氨酸残基与天然酶相比，围绕χ1旋转180度而改变构象。天然蛋白和突变蛋白结构之间FAD辅因子的异咯嗪环部分的比较显示，从非平面几何结构变为平面几何结构（导致N5具有更类似四面体的几何结构）。这种变化被认为是导致观察到的氧化还原电位改变的主要因素。由于在其他共价黄素蛋白中未观察到黄素的类似扭曲，因此提出这代表了一种促进共价胆固醇氧化酶中黄素还原的特定模式。
