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Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase.

ABSTRACT: Horseradish peroxidase (HRP), has gained significant interests in biotechnology, especially in biosensor field and diagnostic test kits. Hence, its solvent-exposed lysine residues 174, 232, and 241 have been frequently modified with the aim of improving its stability and catalytic efficiency. In this computational study, we investigated the effects of Lys-to-Glu substitutions on HRP structure to model charge-reversal manipulations at the enzyme surface. Simulation results implied that upon these substitutions, the number of stable hydrogen bonds and ?-helical content of HRP are increased and the proximal Ca2+ binding pocket becomes more integrated. The results revealed that although Glu174-heme hydrogen bond is lost after mutation, formation of a new hydrogen bonding network contributes to the stability of heme-protein linkage. Together, it may be concluded that these substitutions enhance the stability of the protein moiety as well as the heme-protein non-covalent interactions. In the enzyme active site, we observed increased accessibility of peroxide binding site and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the bottleneck entry of the peroxide-binding site has become wider and more flexible upon substitutions. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is more extended in mutated enzyme. These observations suggest that the reactivity of the enzyme to its substrates has increased. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved upon manipulation of charge distribution on protein surface.

SUBMITTER: Navapour L

PROVIDER: S-EPMC5219912 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase.

Navapour Leila L Mogharrab Navid N

Molecular biology research communications 20160901 3

Horseradish peroxidase (HRP), has gained significant interests in biotechnology, especially in biosensor field and diagnostic test kits. Hence, its solvent-exposed lysine residues 174, 232, and 241 have been frequently modified with the aim of improving its stability and catalytic efficiency. In this computational study, we investigated the effects of Lys-to-Glu substitutions on HRP structure to model charge-reversal manipulations at the enzyme surface. Simulation results implied that upon these ...[more]

PMID: 28097171

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Project description:BackgroundHorseradish Peroxidase (HRP) plays important roles in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Often, it is used in immobilised form. With conventional immobilisation techniques, the enzyme adheres in random orientation: the active site may face the solid phase rather than bulk medium, impeding substrate access and leading to sub-optimal catalytic performance. The ability to immobilise HRP in a directional manner, such that the active site would always face outwards from the insoluble matrix, would maximise the immobilised enzyme's catalytic potential and could increase HRP's range of actual and potential applications.ResultsWe have replaced arginine residues on the face of glycan-free recombinant HRP opposite to the active site by lysines. Our strategy differs from previous reports of specific HRP immobilisation via an engineered affinity tag or single reactive residue. These conservative Arg-to-Lys substitutions provide a means of multipoint covalent immobilisation such that the active site will always face away from the immobilisation matrix. One triple and one pentuple mutant were generated by substitution of solvent-exposed arginines on the "back" of the polypeptide (R118, R159 and R283) and of residues known to influence stability (K232 and K241). Orientated HRP immobilisation was demonstrated using a modified polyethersulfone (PES) membrane; the protein was forced to orientate its active site away from the membrane and towards the bulk solution phase. Mutant properties and bioinformatic analysis suggested the reversion of K283R to improve stability, thus generating two additional mutants (K118/R159K and R118K/K232N/K241F/R283K). While most mutants were less stable in free solution than wild type rHRP, the quadruple revertant regained some stability over its mutant counterparts. A greater degree of immobilisation on CNBr-activated Sepharosetrade mark was noted with increased lysine content; however, only marginal gains in solvent stability resulted from immobilisation on this latter matrix.ConclusionDirectional, orientated, immobilisation of rHRP mutants onto an activated, modified polyethersulfone membrane has been achieved with excellent retention of catalytic activity; however, re-engineering of acceptable stability characteristics into the "immobilisation mutants" will determine their applicability in diagnosis and biosensor development.

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.

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Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase.

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Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase.

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