Autocatalytic formation of a hydroxy group at Cβ of Trp171 in lignin peroxidase
Autocatalytic formation of a hydroxy group at Cβ of Trp171 in lignin peroxidase
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DOI：
10.1021/bi9727186
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发表时间：
1998-06-23
期刊：
影响因子：
2.9
通讯作者：
Piontek, K
中科院分区：
文献类型：
--
作者：
Blodig, W;Doyle, WA;Piontek, K
在来自白腐真菌 Phanerochaete chrysosporium 的两种木质素过氧化物酶同工酶的高分辨率晶体结构中，在距 Trp171 的 C β 的单键距离处观察到显着的电子密度，并将其解释为羟基。为了进一步阐明这一特征的性质，我们对该酶进行了胰蛋白酶消化并分离出了含有Trp171的肽。在环境条件下，该肽显示出色氨酸的典型吸收光谱。然而，在升高的温度下，可以形成 lambda(max) = 333 nm 的不寻常吸收光谱，该吸收光谱与 N-乙酰基-α,β-二脱氢色氨酸酰胺的吸收光谱相同，这是从 β-羟基色氨酸中消除水时产生的。从大肠杆菌产生的重组和重折叠木质素过氧化物酶中分离出的含有 Trp171 的胰蛋白酶肽在任何温度下都不包含特征性 333 nm 吸收带。然而，用 3 当量的 H2O2 处理会导致 Trp171 完全羟基化，减少底物与该过程的竞争，例如，在 0.5 mM 藜芦醇存在下，大约需要 7 当量的 H2O2 才能完成修饰。我们得出结论，Trp 171 C beta 处的羟基化是一种自催化反应，在自然周转的条件下很容易发生，例如，在 P、chrysosporium 的木质素分解培养物中，已知其含有基于氧化酶的 H2O2 生成系统。未发现该氧化过程对分子氧的依赖。用色氨酸特异性试剂 N-溴琥珀酰亚胺对真菌木质素过氧化物酶进行化学修饰，导致其对底物藜芦醇的活性急剧降低。这表明 Trp171 参与催化作用，并且在稳态条件下电子从该表面残基转移到氧化血红素辅因子是可能的。
In the high-resolution crystal structures of two lignin peroxidase isozymes from the white rot fungus Phanerochaete chrysosporium a significant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group. To further clarify the nature of this feature, we carried out tryptic digestion of the enzyme and isolated the Trp171 containing peptide. Under ambient conditions, this peptide shows an absorbance spectrum typical of tryptophan, At elevated temperature, however, the formation of an unusual absorbance spectrum with lambda(max) = 333 nm can be followed that is identical to that of N-acetyl-alpha,beta-didehydrotryptophanamide, resulting upon water elimination from beta-hydroxy tryptophan. The Trp171 containing tryptic peptide isolated from the recombinant and refolded lignin peroxidase produced from Escherichia coli does not contain the characteristic 333 nm absorbance band at any temperature. However, treatment with 3 equiv of H2O2 leads to complete hydroxylation of Trp171, Reducing substrates compete with this process, e.g., in the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for complete modification. We conclude that the hydroxylation at the C beta of Trp 171 is an autocatalytic reaction which occurs readily under conditions of natural turnover, e.g., in the ligninolytic cultures of P, chrysosporium, which are known to contain an oxidase-based H2O2-generating system. No dependence on dioxygen was found for this oxidative process, Chemical modification of fungal lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads to a drastically reduced acitivity with respect to the substrate veratryl alcohol. This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady-state conditions.