2VKA

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

Summary for 2VKA

• Entry DOI: 10.2210/pdb2vka/pdb
• Related: 2BOQ
• Descriptor: VERSATILE PEROXIDASE VPL2, SULFATE ION, CALCIUM ION, ... (6 entities in total)
• Functional Keywords: lignin peroxidase, lignin degradation, manganese peroxidase, polyvalent peroxidase, aromatic-substrate binding, mn-independent oxidation, oxidoreductase
• Biological source: PLEUROTUS ERYNGII (THISTLE MUSHROOM)
• Cellular location: Secreted: O94753
• Total number of polymer chains: 1
• Total formula weight: 34219.07

Authors

Ruiz-Duenas, F.J.,Morales, M.,Mate, M.J.,Romero, A.,Martinez, M.J.,Smith, A.,Martinez, A.T. (deposition date: 2007-12-18, release date: 2008-01-29, Last modification date: 2024-10-16)

Primary citation

Ruiz-Duenas, F.J.,Morales, M.,Mate, M.J.,Romero, A.,Martinez, M.J.,Smith, A.,Martinez, A.T.
Site-Directed Mutagenesis of the Catalytic Tryptophan Environment in Pleurotus Eryngii Versatile Peroxidase
Biochemistry, 47:1685-, 2008

PubMed Abstract: Lignin degradation by fungal peroxidases is initiated by one-electron transfer to an exposed tryptophan radical, a reaction mediated by veratryl alcohol (VA) in lignin peroxidase (LiP). Versatile peroxidase (VP) differs not only in its oxidation of Mn2+ at a second catalytic site but also in its ability to directly oxidize different aromatic compounds. The catalytic tryptophan environment was compared in LiP 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 kinetics of the high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating that other residues contribute to substrate oxidation by the Trp164 radical. Introducing acidic residues at the tryptophan environment did not increase the efficiency of VP oxidizing VA. On the contrary, all variants harboring the R257D mutation lost their activity on RB5. Interestingly, this activity was restored when VA was added as a mediator, revealing the LiP-type behavior of this variant. Moreover, combination of the A260F and R257A mutations strongly increased (20-50-fold) the apparent second-order rate constants for reduction of 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. Nonexposed residues in the vicinity of Trp164 can also affect VP activity, as found with the M247F mutation. This was a direct effect since no modification of the surrounding residues was found in the crystal structure of this variant.

PubMed: 18201105
DOI: 10.1021/BI7020298

Experimental method

X-RAY DIFFRACTION (2 Å)