Chemogenetic Evolution of a Peroxidase-like Artificial Metalloenzyme

Directed evolution has helped enzyme engineering to remarkable successes in the past. A main challenge in directed evolution is to find the most suitable starting point, that is, an enzyme that allows maximum evolvability. Consisting of a synthetic cofactor embedded in a protein scaffold, artificial metalloenzymes (ArMs) are reminiscent of rough-hewn ancestral metalloproteins and thus could provide an evolutionarily clean slate. Here, we report the design and directed evolution of an ArM with peroxidase-like properties based on the nitrobindin variant, NB4. After identifying a suitable artificial metal cofactor, two rounds of directed evolution were sufficient to elevate the ArM's activity to levels akin to those of some natural peroxidases (up to k(cat) = 14.1 s(-1) and k(cat) /K-m= 52,800 M-1 s(-)(1)). A substitution to arginine in the distal cofactor environment (position 76) was the key to boost the peroxidase activity. Molecular dynamics simulations reveal a remarkable flexibility in the distal site of the NB4 scaffold that is absent in the nitrobindin wildtype and which allows the unrestricted movement of the catalytically important Arg76. In addition to the oxidation of the common redox mediators (ABTS, syringaldehyde, and 2,6-dimethoxyphenol), the ArM proved efficient in the decolorization of three recalcitrant dyes (indigo carmine, reactive blue 19, and reactive black 5) and was amenable to several rounds of ArM recycling.

Automated summary

Directed evolution has been successful in enhancing enzyme engineering. In this study, an ArM with peroxidase-like properties was designed and evolved using the nitrobindin variant, NB4. Key to boosting peroxidase activity was a substitution to arginine in the distal cofactor environment. The ArM showed high efficiency in decolorization of dyes and could be recycled multiple times.