Stability properties of an ancient plant peroxidase

• An ancestral plant peroxidase has been cloned and expressed in Escherichia coli.
• It is a recent common ancestor of widely-used horseradish and soybean peroxidases.
• It is estimated to be approx. 110 million year old.
• It is less thermally stable than extant horseradish peroxidase C [HRPC].
• It is more stable to oxidation than extant horseradish and soybean peroxidases.

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.