Mattevi A, Fraaije M W, Mozzarelli A, Olivi L, Coda A, van Berkel W J
Department of Genetics & Microbiology, University of Pavia, Italy.
Structure. 1997 Jul 15;5(7):907-20. doi: 10.1016/s0969-2126(97)00245-1.
Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong impression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkyphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis.
The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elongated cavity on the si side of the flavin molecule. Three residues, Tyr108, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme.
The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.
木质素降解会产生多种芳香族分子，各种真菌微生物可将其作为唯一碳源利用。简单青霉在酚类化合物上生长时，会强烈诱导一种含黄素的香草醇氧化酶（VAO）的产生。该酶催化从芳香胺到4 - 烷基酚等多种底物的氧化反应。VAO是一类广泛分布的新型氧化还原酶成员，其使用黄素腺嘌呤二核苷酸（FAD）作为与蛋白质共价结合的辅因子。我们开展了VAO结构的测定工作，以阐明这些新型氧化还原酶的一些最有趣的特征，比如共价黄素化的功能意义及催化机制。
已测定了天然状态下以及与四种抑制剂形成复合物状态下的VAO晶体结构。该酶是具有42对称性的八聚体；抑制剂结合在黄素分子si侧的一个疏水、细长的腔中。三个残基，即Tyr108、Tyr503和Arg504形成一个阴离子结合亚位点，可稳定底物的酚盐形式。与抑制剂4 - （1 - 庚烯基）酚形成复合物的VAO结构表明，催化腔被该抑制剂完全填满，这解释了为何带有碳链长度超过七个碳原子的脂肪族取代基的烷基酚不能与该酶结合。
活性位点腔的形状通过提供一种“尺寸排阻机制”来控制底物特异性。在腔内，底物芳香环与黄素环呈18度角定位。这种排列非常适合氢化物转移反应，底物去质子化进一步促进了该反应。将底物埋于蛋白质表面之下是许多通过氢化物转移实现底物氧化或还原的黄素酶共有的一种常见策略。
