Chemogenetic Evolution of a Peroxidase-like Artificial Metalloenzyme

Abstract

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.

Learning Epistasis and Residue Coevolution Patterns: Current Trends and Future Perspectives for Advancing Enzyme Engineering

“…Although the high-throughput screening techniques have significantly improved the size of directed-evolution libraries that can be screened, a big problem remains the prediction of and navigation along evolutionary trajectories. On the one hand, due to its mutagenetic constraint to cover only narrow patches of sequence space, the variants generated and cataloged in the library highly depend on the choice of the starting sequences . On the other hand, the progression from each generation of the library to the next relies on an understanding of how to properly combine improved substitutions .…”

“…On the one hand, due to its mutagenetic constraint to cover only narrow patches of sequence space, the variants generated and cataloged in the library highly depend on the choice of the starting sequences. 48 On the other hand, the progression from each generation of the library to the next relies on an understanding of how to properly combine improved substitutions. 4 State-of-the-art experimental approaches often compensate for this problem by relying on libraries with high sequence diversity or sequence saturation approaches.…”

How Does Surface Charge Engineering of Bacillus subtilis Lipase A Improve Ionic Liquid Resistance? Lessons Learned from Molecular Dynamics Simulations

“…Protein engineering emerges as a powerful tool to optimize enzymes to achieve the desired properties. − Charge engineering/modification on enzymes has been shown to be a promising approach to tailoring the enzyme resistance in ILs. ,− In this regard, Nordwald et al fine-tuned the positive-to-negative charged amino acid ratio of chymotrypsin (from bovine pancreas) and observed an increased half-life (1.6- and 4.3-fold) relative to wild-type chymotrypsin in 1-butyl-3-methylimidazolium chloride ([BMIM]­Cl, 20 wt %) and 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM]­[EtSO 4 ], 20 wt %), respectively . Moreover, half-lives of Candida rugosa lipase and Carica papaya papain in [BMIM]Cl were also enhanced by applying the similarly charged amino acid ratio strategy …”

The Role of Glycerol in Preserving Proteins Needs to Be Reconsidered

“…Although these studies have made significant progress in the mechanistic understanding of protein–protectant systems, the inconsequential statements and reasoning indicated that further studies were still required. Also, more and more studies proved the importance of quantitative experimental evidence to compare with theoretical and molecular dynamics (MD) simulation results. − …”