Manganese Oxidation Site in Pleurotus eryngii Versatile Peroxidase: A Site-Directed Mutagenesis, Kinetic, and Crystallographic Study

详细信息    查看全文

• 作者：Francisco J. Ruiz-Due&ntilde ; as ; Marí ; a Morales ; Marta Pé ; rez-Boada ; Thomas Choinowski ; Marí ; a Jesú ; s Martí ; nez ; Klaus Piontek ; Á ; ngel T. Martí ; nez
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：January 9, 2007
• 年：2007
• 卷：46
• 期：1
• 页码：66 - 77
• 全文大小：724K
• 年卷期：v.46,no.1(January 9, 2007)
• ISSN：1520-4995

文摘

The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophanresponsible for aromatic substrate oxidation and a putative Mn²⁺ oxidation site. The crystal structures(solved up to 1.3 Å) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure toMn²⁺, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175,contribute to Mn²⁺ coordination. To evaluate the involvement of these residues, site-directed mutagenesiswas performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn²⁺ affinityand a decrease in the Mn²⁺ oxidation activity. Transient-state kinetic constants showed that reduction ofboth compounds I and II was affected (80-325-fold lower k_2app and 10³-10⁴-fold lower k_3app, respectively).The single mutants retained partial Mn²⁺ oxidation activity, and a triple mutation (E36A/E40A/D175A)was required to completely suppress the activity (<1% k_cat). The affinity for Mn²⁺ also decreased (~25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in k_cat in thecase of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showedthat introduction of a basic residue near Asp175 did not improve Mn²⁺ oxidation as found for MnP andruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn²⁺. Thestructural and kinetic data obtained highlighted significant differences in the Mn²⁺ oxidation site of thenew versatile enzyme compared to P. chrysosporium MnP.