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.

中文翻译：

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Sphingobacterium sp。T2锰超氧化物歧化酶通过生成羟基自由基催化聚合木质素的氧化脱甲基

Sphingobacterium sp。T2包含两个胞外锰超氧化物歧化酶的酶表现出对木质素氧化但前所未有活性通过未知的机理。木质素模型化合物的酶处理产生的产品结构指示芳基-Cα氧化裂解和去甲基化，以及烯烃二羟基化和醇氧化。SpMnSOD催化木质素模型化合物桂酰基甘油-β-愈创木脂醚氧化的¹⁸ O标记研究表明，该酶插入的氧原子衍生自超氧化物或过氧化物。SpMnSOD1处理的碱性木质素的定量分析³¹P NMR光谱显示酚和脂肪族OH含量增加20-40％，与木质素脱甲基化和一些内部氧化裂解反应一致。使用荧光羟苯基荧光素测定法进行的羟基自由基生成分析表明，SpMnSOD1释放了4.1摩尔当量的羟基自由基。研究了SpMnSOD1中的四个氨基酸置换，根据³¹ P NMR分析，发现A31H或Y27H定点突变酶没有木质素脱甲基活性。A31H和Y27H突变酶的结构确定揭示了N末端蛋白环的重新定位，导致二聚体界面上溶剂通道的加宽，这将增加溶剂进入Mn中心的羟基自由基的产生。