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首页> 外文期刊>Biochemistry >Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L
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Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L

机译：藜芦醇木质素过氧化物酶氧化：突变体H82A，Q222A，W171A和F267L的影响

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摘要

The site-directed mutations H82A and Q222A (residues near the heme access channel), and W171A and F267L (residues near the surface of the protein) were introduced into the gene encoding lignin peroxidase (LiP) isozyme H8 from Phanerochaete chrysosporium. The variant enzymes were produced by homologous expression in P. chrysosporium, purified to homogeneity, and characterized by kinetic and spectroscopic methods. The molecular masses, the pIs, and the UV-vis absorption spectra of the ferric and oxidized states of these LiP variant enzymes were similar to those of wild-type LiP (wtLiP), suggesting the overall protein and heme environments were not significantly affected by these mutations. The steady-state and transient-state parameters for the oxidation of veratryl alcohol (VA) by the H82A and Q222A variants were very similar to those of wtLiP, demonstrating that these residues are not involved in VA oxidation and that the heme access channel is an unlikely site for VA oxidation. In contrast, the W171A variant was unable to oxidize VA, confirming the apparent essentiality of Trp171 in VA oxidation by LiP. The kinetic rates of spontaneous LiP compound I reduction in the absence of VA were similar for W171A and wild-type LiP, suggesting that there may not be a radical formed on the Trp171 residue of LiP in the absence of VA. For the F267L variant, both the K_(m app) value in the steady state and the apparent dissociation constant (K_D) for compound II reduction were greater than those for wtLiP. These results indicate that the site including W171 and F267, rather than the heme access channel, is the site of VA binding and oxidation in LiP. Whereas Trp171 appears to be essential for VA oxidation, it apparently is not independently responsible for the spontaneous decomposition of oxidized intermediates. The nearby Phe267 apparently is also involved in VA binding.
机译：将定点突变H82A和Q222A（血红素通道附近的残基）以及W171A和F267L（蛋白表面附近的残基）引入到来自Phanerochaete chrysosporium的木质素过氧化物酶（LiP）同工酶H8的基因中。变异酶通过在金孢假单胞菌中同源表达而产生，纯化至均一并通过动力学和光谱法表征。这些LiP变异酶的三价铁和氧化态的分子量，pIs和UV-vis吸收光谱与野生型LiP（wtLiP）相似，表明整体蛋白质和血红素环境不受显着影响这些突变。 H82A和Q222A变体氧化藜芦醇（VA）的稳态参数和瞬态参数与wtLiP非常相似，表明这些残基不参与VA氧化，并且血红素通道是不太可能发生VA氧化。相反，W171A变体无法氧化VA，从而证实了Trp171在LiP氧化VA中的明显必要性。对于W171A和野生型LiP，在不存在VA的情况下自发LiP化合物I还原的动力学速率相似，这表明在不存在VA的情况下，LiP的Trp171残基上可能没有形成自由基。对于F267L变体，化合物II还原时的稳态K_（m app）值和表观解离常数（K_D）均大于wtLiP。这些结果表明，包括W171和F267在内的部位，而不是血红素通道，是VA在LiP中结合和氧化的部位。尽管Trp171对于VA氧化似乎必不可少，但它显然不是氧化中间体的自发分解的独立原因。附近的Phe267显然也参与了VA结合。

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