Spectroscopic evidence for an engineered catalytically-active Trp radical that creates the unique reactivity of lignin peroxidase

Smith, Andrew T, Doyle, Wendy A, Dorlet, Pierre and Ivancich, Anabella (2009) Spectroscopic evidence for an engineered catalytically-active Trp radical that creates the unique reactivity of lignin peroxidase. Proceedings of the National Academy of Sciences of the United States of America (PNAS), 106 (38). pp. 16084-16089. ISSN 1091-6490

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Abstract

The surface oxidation site (Trp171) in lignin peroxidase (LiP) which is specific for the reaction with the high redox potential substrate, veratryl alcohol (1.4 V), had been previously engineered in a heme peroxidase that has similar protein fold but lacks this activity. The new Trp site was engineered by introducing a Trp residue in the coprinus cinereus peroxidase (CiP) at the equivalent position to that of the Trp171 in LiP. In order to induce the catalytic activity towards veratryl alcohol in CiP, it was also necessary to reproduce the naturally-occurring negatively-charged microenvironment of the Trp site. Multifrequency EPR spectroscopy characterization of the D179W+R258E+R272D variant of CiP unequivocally showed that, upon reaction of the enzyme with hydrogen peroxide a new Trp radical (g-values of gx = 2.0035(5), gy = 2.0027(5) and gz = 2.0022(1)) was formed after the [Fe(IV)=O Por+] intermediate, as a result of intramolecular electron transfer between Trp179 and the porphyrin. The EPR characterization crucially showed that the [Fe(IV)=O Trp] species was the reactive intermediate with veratryl alcohol. Accordingly, our work shows that it is necessary to take into account the physicochemical properties of the radical, fine-tuned by the microenvironment as well as those of the preceding [Fe(IV)=O Por+] intermediate to engineer a catalytically-competent Trp site for a given substrate. This work also highlights the role of Trp as a tunable redox-active cofactor for enzyme catalysis, in the context of peroxidases with a unique reactivity towards recalcitrant substrates that require oxidation potentials not attainable by the heme cofactor

Item Type: Article
Schools and Departments: School of Life Sciences > Biochemistry
Depositing User: Andrew Trevor Smith
Date Deposited: 31 Jan 2013 13:52
Last Modified: 31 Jan 2013 13:53
URI: http://sro.sussex.ac.uk/id/eprint/16465
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