Site-Directed Mutagenesis of the Catalytic Tryptophan Environment in Pleurotus eryngii Versatile Peroxidase

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• 作者：Francisco J. Ruiz-Dueñ ; as ; Mar&iacute ; a Morales ; Mar&iacute ; a J. Mate ; Antonio Romero ; Mar&iacute ; a Jesú ; s Mart&iacute ; nez ; Andrew T. Smith ; Á ; ngel T. Mart&iacute ; nez
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：February 12, 2008
• 年：2008
• 卷：47
• 期：6
• 页码：1685 - 1695
• 全文大小：422K
• 年卷期：v.47,no.6(February 12, 2008)
• ISSN：1520-4995

文摘

Lignin degradation by fungal peroxidases is initiated by one-electron transfer to an exposedtryptophan radical, a reaction mediated by veratryl alcohol (VA) in lignin peroxidase (LiP). Versatileperoxidase (VP) differs not only in its oxidation of Mn²⁺ at a second catalytic site but also in its abilityto directly oxidize different aromatic compounds. The catalytic tryptophan environment was compared inLiP and VP crystal structures, and six residues near VP Trp164 were modified by site-directed mutagenesis.Oxidation of Mn²⁺ was practically unaffected. However, several mutations modified the oxidation kineticsof the high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating that other residuescontribute to substrate oxidation by the Trp164 radical. Introducing acidic residues at the tryptophanenvironment did not increase the efficiency of VP oxidizing VA. On the contrary, all variants harboringthe R257D mutation lost their activity on RB5. Interestingly, this activity was restored when VA wasadded as a mediator, revealing the LiP-type behavior of this variant. Moreover, combination of the A260Fand R257A mutations strongly increased (20-50-fold) the apparent second-order rate constants for reductionof VP compounds I and II by VA to values similar to those found in LiP. Dissociation of the enzyme-product complex seemed to be the limiting step in the turnover of this improved variant. Nonexposedresidues in the vicinity of Trp164 can also affect VP activity, as found with the M247F mutation. Thiswas a direct effect since no modification of the surrounding residues was found in the crystal structure ofthis variant.