Blodig W, Doyle W A, Smith A T, Winterhalter K, Choinowski T, Piontek K
Laboratory of Biochemistry I, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
Biochemistry. 1998 Jun 23;37(25):8832-8. doi: 10.1021/bi9727186.
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 Trp171 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 activity 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.
在来自白腐真菌黄孢原毛平革菌的两种木质素过氧化物酶同工酶的高分辨率晶体结构中，观察到在距Trp171的Cβ单键距离处有显著的电子密度，并将其解释为一个羟基。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，并分离出含Trp171的肽段。在环境条件下，该肽段显示出典型的色氨酸吸收光谱。然而，在升高的温度下，可以观察到形成了一种不寻常的吸收光谱，其最大吸收波长λmax = 333 nm，这与β-羟基色氨酸脱水后生成的N-乙酰-α,β-二脱氢色氨酸酰胺的吸收光谱相同。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Trp171的胰蛋白酶肽段在任何温度下都不含有特征性的333 nm吸收带。然而，用3当量的H2O2处理会导致Trp171完全羟基化。还原底物会与这一过程竞争，例如，在存在0.5 mM藜芦醇的情况下，完全修饰大约需要7当量的H2O2。我们得出结论，Trp171的Cβ羟基化是一种自催化反应，在自然周转条件下很容易发生，例如在黄孢原毛平革菌的木质素分解培养物中，已知该培养物含有基于氧化酶的H2O2生成系统。未发现该氧化过程对双原子氧有依赖性。用色氨酸特异性试剂N-溴代琥珀酰亚胺对真菌木质素过氧化物酶进行化学修饰，会导致其对底物藜芦醇的活性大幅降低。这表明Trp171参与催化作用，并且在稳态条件下，该表面残基向氧化的血红素辅因子的电子转移是可能的。
