Sphingobacterium sp. T2 Manganese Superoxide Dismutase Catalyzes the Oxidative Demethylation of Polymeric Lignin via Generation of Hydroxyl Radical.
Sphingobacterium sp. T2 Manganese Superoxide Dismutase Catalyzes the Oxidative Demethylation of Polymeric Lignin via Generation of Hydroxyl Radical.
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DOI：
10.1021/acschembio.8b00557
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发表时间：
2018-09
影响因子：
4
通讯作者：
G. M. Rashid;Xiaoyang Zhang;R. Wilkinson;V. Fülöp;B. Cottyn;S. Baumberger;T. Bugg
中科院分区：
文献类型：
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作者：
G. M. Rashid;Xiaoyang Zhang;R. Wilkinson;V. Fülöp;B. Cottyn;S. Baumberger;T. Bugg
鞘氨醇杆菌属。T2含有两个胞外的锰超氧化物歧化酶，它们对木质素的氧化表现出前所未有的活性，但机制未知。木素模型化合物的酶处理产物具有芳基C-α氧化裂解和脱甲基化、烯烃二羟基化和醇氧化等结构特征。~(18)O标记研究表明，木素模型化合物愈创木基甘油-β-愈创木基醚催化氧化反应中的氧原子来源于超氧化物或过氧化氢。经SpMnSOD1处理的碱木素的31P核磁共振定量分析表明，酚类和脂肪族羟基的含量增加了20-40%，这与木质素去甲基化和一些内部氧化裂解反应一致。羟基苯基荧光素荧光测定法测定羟基自由基释放量为4.1摩尔当量的羟基自由基。对SpMnSOD1中的4个氨基酸替换进行了研究，经31P核磁共振分析发现，A31H或Y27H定点突变酶不显示木质素去甲基化活性。对A31H和Y27H突变酶的结构测定表明，N-末端蛋白质环重新定位，导致二聚体界面上的溶剂通道加宽，这将为羟基自由基的产生提供更多的溶剂进入Mn中心。
Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-Cα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-β-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation.