Shrestha Ruben, Chen Xuejie, Ramyar Kasra X, Hayati Zahra, Carlson Eric A, Bossmann Stefan H, Song Likai, Geisbrecht Brian V, Li Ping
Department of Chemistry, Kansas State University, Manhattan, KS, 66506, USA.
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, 66506, USA.
ACS Catal. 2016 Dec 2;6(12):8036-8047. doi: 10.1021/acscatal.6b01952. Epub 2016 Oct 12.
Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases, in which a catalytic distal aspartate is involved in HO activation to catalyze oxidations in acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial DyP from is among the most active DyPs and shows activity toward phenolic lignin model compounds (, , 23447). Based on the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II-like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the DyP catalyzes oxidation of large-size substrates multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in DyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the crosslinking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class DyP is minimal possibly due to its extremely small solvent accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.
染料脱色过氧化物酶（DyPs）是血红素过氧化物酶家族的成员，其中催化性远端天冬氨酸参与过氧化氢的活化，以催化酸性条件下的氧化反应。由于它们在木质素化合物降解和生物质生物燃料生产中的潜在应用，受到了广泛关注。然而，细菌DyPs的氧化模式仍然未知。我们最近报道，来自[具体来源未给出]的细菌DyP是活性最高的DyPs之一，并且对酚类木质素模型化合物（[列举的具体化合物未给出]）具有活性。基于在1.75 Å分辨率下解析的X射线晶体结构、与活性蓝19（RB19）的S型稳态动力学，以及通过停流光谱和电子顺磁共振（EPR）在没有还原底物的情况下观察到的类似化合物II产物的形成，我们推测DyP通过多个表面暴露的蛋白质自由基催化大尺寸底物的氧化。在由376个残基组成的成熟蛋白质的DyP中的7个色氨酸和3个酪氨酸中，W263、W376和Y332被鉴定为表面暴露的蛋白质自由基。只有W263也被表征为表面暴露的氧化位点之一。SDS-PAGE和尺寸排阻色谱表明，W376代表电子转移的一条非主要途径的终点，导致在没有底物的情况下蛋白质交联。W376的突变提高了化合物I的稳定性以及对RB19的整体催化效率。虽然Y332在所有四类DyPs中高度保守，但其在A类DyP中的催化功能可能由于其极小的溶剂可及面积而最小。鉴定表面暴露的蛋白质自由基和底物氧化位点对于理解DyP的机制以及调节其催化功能以提高对酚类木质素的活性很重要。
