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Abstract：
BACKGROUND: Manganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure-function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule.
RESULTS: Two MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD-referred to as Ape-MnP1 and Cst-MnP1, respectively-were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation.
CONCLUSIONS: Understanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure-function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.
RESULTS: Two MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD-referred to as Ape-MnP1 and Cst-MnP1, respectively-were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation.
CONCLUSIONS: Understanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure-function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.
摘要：
背景：锰过氧化物酶（MnPs）是，与木质素过氧化物酶和通用过氧化物酶一起，白腐真菌分泌的酶促机制的关键元素降解木质素，从而在植物细胞壁中提供获得纤维素和半纤维素的途径。最近对52种蘑菇物种的基因组分析显示，存在新的MnP亚家族，这些亚家族在构成锰氧化位点的氨基酸残基上有所不同。在这个模拟分析之后，需要进行全面的结构-功能研究，以了解这些酶是如何工作的，并有助于转化木质素大分子。
结果：属于最近分类为MnP-DGD和MnP-ESD的亚家族的两个MnP-分别称为Ape-MnP1和Cst-MnP1-被鉴定为主要的过氧化物酶。在木质纤维素底物上生长时，Agaricales物种Agrocubepiades和Cyathusstriatus分泌的。异源表达和体外激活后，它们的生化特性证实这些酶是有活性的MnPs。然而,晶体结构和诱变研究表明，锰配位球与初始分类后的预期不同。具体来说,发现Ape-MnP1的C末端尾部的谷氨酰胺残基（Gln333）与锰结合有关，与Asp35和Asp177一起，而Cst-MnP1仅计数两种氨基酸（Glu36和Asp176），而不是三个，起到MnP的作用。这些发现导致将这些亚家族重命名为MnP-DDQ和MnP-ED，并重新评估其进化起源。两种酶都能够直接氧化木质素衍生的酚类化合物，如其他短MnP所示。重要的是,尺寸排阻色谱分析表明，在锰的存在下，两种酶都会引起聚合木质素的变化，表明它们在木质纤维素转化中的相关性。
结论：了解担子菌降解木质素的机制对于理解自然界中的碳循环和设计用于植物生物质工业用途的生物技术工具特别重要。这里,我们提供了蘑菇中存在的两个新的MnP亚家族的第一个结构-功能表征，阐明参与催化的主要残基，并证明它们修饰木质素大分子的能力。
结果：属于最近分类为MnP-DGD和MnP-ESD的亚家族的两个MnP-分别称为Ape-MnP1和Cst-MnP1-被鉴定为主要的过氧化物酶。在木质纤维素底物上生长时，Agaricales物种Agrocubepiades和Cyathusstriatus分泌的。异源表达和体外激活后，它们的生化特性证实这些酶是有活性的MnPs。然而,晶体结构和诱变研究表明，锰配位球与初始分类后的预期不同。具体来说,发现Ape-MnP1的C末端尾部的谷氨酰胺残基（Gln333）与锰结合有关，与Asp35和Asp177一起，而Cst-MnP1仅计数两种氨基酸（Glu36和Asp176），而不是三个，起到MnP的作用。这些发现导致将这些亚家族重命名为MnP-DDQ和MnP-ED，并重新评估其进化起源。两种酶都能够直接氧化木质素衍生的酚类化合物，如其他短MnP所示。重要的是,尺寸排阻色谱分析表明，在锰的存在下，两种酶都会引起聚合木质素的变化，表明它们在木质纤维素转化中的相关性。
结论：了解担子菌降解木质素的机制对于理解自然界中的碳循环和设计用于植物生物质工业用途的生物技术工具特别重要。这里,我们提供了蘑菇中存在的两个新的MnP亚家族的第一个结构-功能表征，阐明参与催化的主要残基，并证明它们修饰木质素大分子的能力。
