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Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation.

ABSTRACT: Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing, and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of this modification, which alters three accessible charged lysines (Lys-174, Lys-232, and Lys-241) to the hydrophobic anthraquinolysine residues. Thermal denaturation and thermoinactivation studies demonstrated that this kind of modification enhances the conformational and operational stability of HRP. The melting temperature increased 3 degrees C and the catalytic efficiency enhanced by 80%. Fluorescence and circular dichroism investigations suggest that the modified HRP benefits from enhanced aromatic packing and more buried hydrophobic patches as compared to the native one. Molecular dynamics simulations showed that modification improves the accessibility of His-42 and the heme prosthetic group to the peroxide and aromatic substrates, respectively. Additionally, the hydrophobic patch, which functions as a binding site or trap for reducing aromatic substrates, is more extended in the modified enzyme. In summary, this modification produces a new derivative of HRP with enhanced electron transfer properties, catalytic efficiency, and stability for biotechnological applications.

SUBMITTER: Mogharrab N

PROVIDER: S-EPMC1783884 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation.

Mogharrab Navid N Ghourchian Hedayatollah H Amininasab Mehriar M

Biophysical journal 20061117 4

Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing, and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of th ...[more]

PMID: 17114227

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Project description:Horseradish Peroxidase (HRP) is one of the most studied peroxidases and a great number of chemical modifications and genetic manipulations have been carried out on its surface accessible residues to improve its stability and catalytic efficiency necessary for biotechnological applications. Most of the stabilized derivatives of HRP reported to date have involved chemical or genetic modifications of three surface-exposed lysines (K174, K232 and K241). In this computational study, we altered these lysines to phenylalanine residues to model those chemical modifications or genetic manipulations in which these positively charged lysines are converted to aromatic hydrophobic residues. Simulation results implied that upon these substitutions, the protein structure becomes less flexible. Stability gains are likely to be achieved due to the increased number of stable hydrogen bonds, improved heme-protein interactions and more integrated proximal Ca2+ binding pocket. We also found a new persistent hydrogen bond between the protein moiety (F174) and the heme prosthetic group as well as two stitching hydrogen bonds between the connecting loops GH and F'F″ in mutated HRP. However, detailed analysis of functionally related structural properties and dynamical features suggests reduced reactivity of the enzyme toward its substrates. Molecular dynamics simulations showed that substitutions narrow the bottle neck entry of peroxide substrate access channel and reduce the surface accessibility of the distal histidine (H42) and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the area and volume of the aromatic-substrate binding pocket are significantly decreased upon modifications. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is shrunk in mutated enzyme. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved concurrent with a decrease in enzyme activity, upon manipulation of charge/hydrophobicity balance at the protein surface.

Project description:BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

Dataset Information

Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation.

Publications

Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation.

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