Caldinelli Laura, Iametti Stefania, Barbiroli Alberto, Bonomi Francesco, Fessas Dimitrios, Molla Gianluca, Pilone Mirella S, Pollegioni Loredano
Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy.
J Biol Chem. 2005 Jun 17;280(24):22572-81. doi: 10.1074/jbc.M500549200. Epub 2005 Apr 7.
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 (approximately 3 versus approximately 5 M, respectively), and the mutant protein had a melting temperature approximately 10-15 degrees 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.
来自短杆菌属的胆固醇氧化酶是一种单体黄素酶，催化胆固醇氧化并异构化为胆甾-4-烯-3-酮。该蛋白属于II类胆固醇氧化酶，FAD辅因子通过His(69)残基与酶共价连接。在这项工作中，将野生型胆固醇氧化酶的去折叠与H69A突变体的去折叠进行了比较，该突变体不与黄素辅因子共价结合。这两种蛋白形式在整体拓扑结构上没有显著差异，但尿素诱导的H69A突变体去折叠发生时的尿素浓度显著低于野生型（分别约为3M和5M），并且在热变性实验中，突变体蛋白的解链温度比野生型低约10-15℃。用于监测蛋白去折叠的各种光谱特征的不同敏感性表明，两种蛋白都发生了两步（三态）过程。在2m尿素浓度下，H69A突变体中中间体的存在更为明显，此时催化活性和三级结构丧失，并且蛋白表面暴露出新的疏水区域，导致蛋白聚集。对野生型和H69A蛋白进行尿素和热处理后发生的变化进行比较分析表明，蛋白不稳定性与黄素和蛋白之间共价连接的消除之间存在良好的相关性。这种共价键代表了一种结构机制，可改变黄素的氧化还原电位并稳定胆固醇氧化酶的三级结构，从而指出了黄素结合模式在致病性和非致病性细菌中进行相同反应的酶中的特定意义。
