Chaplin Amanda K, Svistunenko Dimitri A, Hough Michael A, Wilson Michael T, Vijgenboom Erik, Worrall Jonathan A R
School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands.
Biochem J. 2017 Feb 20;474(5):809-825. doi: 10.1042/BCJ20160968.
GlxA from is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best characterised. GlxA is a key cuproenzyme in the copper-dependent morphological development of with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic sites in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper-co-ordinating tyrosine ligand and houses a radical. In GlxA and Gox, a second co-ordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes, creating a marked difference in the π-π stacking interaction of the benzyl ring with the 3'-(-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here, we report a series of and studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development.
来自[具体来源未提及]的GlxA是一种单核铜自由基氧化酶，属于辅助活性家族5（AA5）的成员。其结构域组织和低序列同源性使其成为AA5家族中一个独特的成员，其中真菌半乳糖6-氧化酶（Gox）是特征最明确的。GlxA是[具体物种未提及]铜依赖性形态发育中的关键铜酶，其功能与胞外聚糖的加工有关。GlxA和Gox中的催化位点包含两个不同的单电子受体，即铜离子和一个3'-（-半胱氨酰）酪氨酸。后者通过半胱氨酸与铜配位酪氨酸配体之间的共价键在翻译后形成，并含有一个自由基。在GlxA和Gox中，存在第二个配位球色氨酸残基（GlxA中的Trp288），但两种酶中吲哚环的取向不同，导致苄基环与3'-（-半胱氨酰）酪氨酸的π-π堆积相互作用存在显著差异。据报道，GlxA和Gox之间在光谱和酶活性方面存在差异，吲哚取向被认为是原因。在这里，我们报告了一系列使用GlxA的W288F和W288A变体进行的[具体实验未明确说明]研究，以评估Trp288对形态、成熟、光谱和酶学性质的作用。我们的研究结果表明，Trp288在GlxA的铜负载和成熟动力学中起着重要作用，其存在对于稳定耦合催化活性和形态发育所需的金属自由基位点至关重要。
