The structure of a mutant enzyme of Coprinus cinereus peroxidase provides an understanding of its increased thermostability

Seven amino‐acid substitutions introduced into the 343 amino‐acid‐long sequence of Coprinus cinereus peroxidase (CiP) led to a mutant enzyme (TS‐rCiP) which is more stable than the native enzyme at higher temperature, pH and hydrogen peroxide concentrations. It is therefore more suitable for industrial applications. A structure determination was conducted on a deglycosylated but still active form of TS‐­rCiP based on X‐ray diffraction data to 2.05 Å resolution measured on a crystal cooled to 100 K and refined to R = 0.202 and Rfree = 0.249. The increased stability of the TS‐rCiP enzyme can be understood from the structural changes of the TS‐rCiP structure revealed by a comparative analysis with other known CiP structures. One of the more significant changes caused by three of the substitutions, I49S, V53A and T121A, is the conversion of a hydrophobic pocket into a hydrophilic pocket with associated changes in the water structure and the hydrogen‐bonding interactions. The E239G substitution, which gives rise to increased thermostability at high pH, creates changes in the water structure and in the orientation of a phenylalanine (Phe236) in its vicinity. The three substitutions M166F, M242 and Y242F introduced to increase the oxidative stability do not introduce any structural changes.