title: Stability properties of an ancient plant peroxidase.
abstract: Plant (Class III) peroxidases have numerous applications throughout biotechnology but their thermal and oxidative stabilities may limit their usefulness. Horseradish peroxidase isoenzyme C (HRPC) has good catalytic turnover and is moderately resistant to heat and to excess (oxidizing) concentrations of hydrogen peroxide. In contrast, HRP isoenzyme A2 (HRP A2) has better oxidative but poorer thermal stability, while soybean peroxidase (SBP) displays enhanced thermal stability. Intrigued by these variations amongst closely related enzymes, we previously used maximum likelihood methods (with application of Bayesian statistics) to infer an amino acid sequence consistent with their most recent common ancestor, the 'Grandparent' (GP). Here, we report the cloning and expression of active recombinant GP protein in Escherichia coli. GP's half-inactivation temperature was 45 °C, notably less than HRP C's, but its resistance to excess H2O2 was 2-fold greater. This resurrected GP protein enables a greater understanding of plant peroxidase evolution and serves as a test-bed to explore their ancestral properties.
authors.affiliation: School of Biotechnology, Dublin City University, Dublin 9, Ireland. Electronic address: noeleen.loughran@gmail.com.
authors.forename: N B
authors.initials: NB
authors.lastname: Loughran
authors.affiliation: School of Biotechnology, Dublin City University, Dublin 9, Ireland. Electronic address: mary.oconnell@dcu.ie.
authors.forename: M J
authors.initials: MJ
authors.lastname: O'Connell
authors.affiliation: School of Biotechnology, Dublin City University, Dublin 9, Ireland. Electronic address: brendan.oconnor@dcu.ie.
authors.forename: B
authors.initials: B
authors.lastname: O'Connor
authors.affiliation: School of Biotechnology, Dublin City University, Dublin 9, Ireland. Electronic address: ciaran.fagan@dcu.ie.
authors.forename: C
authors.initials: C
authors.lastname: O'Fágáin
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000187:drug effects; D019143:Evolution, Molecular; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D013696:Temperature
chemical_list: D006861:Hydrogen Peroxide; D009195:Peroxidase
keywords: Ancestral protein; Oxidative stability; Plant peroxidase; Thermal stability