Abstract
Versatile peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved.
Highlights
Lignin degradation has been a hot topic of research for several decades, and still actual nowadays
Isopropyl-β-D-thiogalactopyranoside (IPTG), dithiothreitol (DTT), hemin, oxidized glutathione (GSSG), veratryl alcohol (VA), manganese(II) sulphate, Reactive Black 5 (RB5), 2,6-dimethoxyphenol (DMP), sodium tartrate and other chemicals were purchased from Sigma-Aldrich; urea and hydrogen peroxide were from Merck; and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) from Roche
P. eryngii Versatile peroxidase (VP) and P. ostreatus MnP4 share a common structural scaffold (Fig 1A). Their crystal structures (PDB entries 2BOQ for VP, and 4BM1 for MnP4) superimpose with a root mean square deviation of only 0.75 Å between the Cα positions over 316 amino acid residues, covering 95% of the mature proteins. This high structural similarity between both proteins was the basis of our strategy aimed to improve the pH stability of VP, which consisted in identifying the stabilizing motifs putatively contributing to the high stability towards pH of MnP4, and their subsequent transfer into VP
Summary
Lignin degradation has been a hot topic of research for several decades, and still actual nowadays. The socalled white-rot fungi belonging to the group of basidiomycetes are unique due to their ability to degrade lignin from plant biomass in an efficient way This process begins with the unspecific oxidative attack to the aromatic units of this polymer by means of a battery of extracellular oxidoreductases among which ligninolytic peroxidases play a key role [2]. VP combines catalytic properties of the above two families due to the presence of both a Mn-oxidation site [13] and a catalytic tryptophan [14] in its molecular structure This peroxidase exhibits characteristics of GPs by its ability to oxidize low redox potential substrates (e.g. phenols) at the main heme access channel [15]
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