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Expression and characterisation of a novel manganese peroxidise from Phlebia radiata

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posted on 2023-06-07, 15:35 authored by Usenobong Friday Ufot
Manganese peroxidase 3 (MnP3) is an extracellular peroxidase from the white-rot fungus, P. radiata that is a well-studied wood/lignin degrader from Scandinavian forest (Hilden et al., 2005). Unlike the Phanerochaete chrysosporium MnP's previously characterised, the P. radiata MnP3 is a 'short' MnP with a truncated C-terminus evolutionarily more related to the newly characterised versatile peroxidase. The cDNA for the full length MnP3 from P. radiata was optimised at the 5' end for E.coli expression and cloned into the expression vector pFLAG1. Five site-directed mutants targetting the Mn binding site (E40H, E44H, E40H/E44H, D186H and D186N) were constructed and characterised. The recombinant MnP3‘s, both wild-type and variants were isolated from E.coli inclusion bodies after expression in W3110 and active soluble proteins recovered after in vitro refolding and extensively characterised. In this work, the characteristics of the P. radiata wild type MnP3 were compared with those of the mutant enzymes and the 'classical' Phanerochaete enzyme described in the literature. The molecular weight of the enzymes was 36kDa with UV/Vis absorption spectra typical of a six-coordinate high spin haem peroxidase. In contrast to the literature on these enzymes, the Phlebia MnP3 was found to have a maximum efficacy (kcat/Km) at pH 5.0 rather than the more typical optimum at 4.5. Consistent with the high oxidation potential of these enzymes, the Compound I form of the enzyme was found to be unstable decaying rapidly to a Compound II-like intermediate. Mutations to the manganese (II)-binding ligands change the catalytic properties of the Phlebia MnP3 dramatically and reactivity towards Mn (II) were greatly decreased particularly at lower pH. Interestingly, much of the activity in the higher pH region was maintained, showing that carboxylate Mn ligands are not essential at higher pH when tighter binding of Mn2+ is favoured. The pKa‘s for the activity dependence matches those expected for the deprotonation of the surrogate His ligands. The results confirm that the mutated Mn (II) binding site is the only productive catalytic site for Mn (II) oxidation. The specificity of metal binding by MnP3 was examined; Cu ions were found to be strongly inhibitory and Zinc ions bound particularly tightly and somewhat nonspecifically to MnP3 with stoichiometries as high as 5:1 suggestive of multiple metal-binding sites other than the specific manganese-binding site. Use of Zn ions in crystallisation media clearly has the potential to complicate metal ion assignment in the structures of these enzymes. Interestingly, Cobalt ions were able to enhance the activity of MnP3 but were not rapidly oxidised by the enzyme itself. The P. radiata enzymes have been found to be active in acidic and alkaline regions, being stable over a large pH range (5.0 – 8.5) and are readily additionally stabilised by addition of excess Ca2+, both desirable features in such an enzyme that could be applied to the 'biorefinery' concept.

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  • Published version

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346.0

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  • Biochemistry Theses

Qualification level

  • doctoral

Qualification name

  • dphil

Language

  • eng

Institution

University of Sussex

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  • Yes

Legacy Posted Date

2010-11-04

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