Exploring the design of copper artificial metalloenzymes Eva Klemencic 1* , Richard Brewster 1 , Hafiz Ali 2 , Julia Richardson 3 , Amanda Jarvis 1 1 EastChem School of Chemistry, University of Edinburgh, UK, 2 INEOS Oxford Institute, University of Oxford, UK, 3 School of Biological Sciences, University of Edinburgh, UK *E.Klemencic@sms.ed.ac.uk Artificial metalloenzymes (ArMs), alongside enzyme engineering and directed evolution, have been part of the effort to introduce new-to-nature reactions of transition metals into the realm of biocatalysis, expanding the biocatalytic toolbox. [1] ArMs combine a protein scaffold, which acts as a secondary coordination sphere around a synthetic metal complex that provides the reactive center. Whilst the protein scaffold can have an intrinsic ability to bind metals, a common approach to ArM design is to incorporate a metal binding moiety to integrate the metal into the scaffold site specifically. Direct comparisons of the same metal ligation environment and reaction using different modification strategies in the field of ArMs have been rare, making it hard to draw conclusions about the best method of modification for a given application. The production of proteins containing unnatural amino acids typically results in lower yields, whilst the post-translational modification of proteins takes extra steps increasing time. In this work, we explored how different modification methods of a protein scaffold would lead to different metalloproteins structures and the role this might play in catalytic activity and selectivity. Research in the Jarvis group has been focused on the design and construction of novel Copper ArMs using steroid carrier protein (SCP) [2] as the protein scaffold and Cu(II) bound to bipyridine (Bpy) as the catalytic center. The Bpy was introduced into SCP using two different strategies; either amber codon expression to incorporate Bpy as an unnatural amino acid (2,2’-bipyridin-5-yl)alanine (BpyAla)[3, 4], or the bioconjugation of a bromoalkyl-tethered bipyridine to cysteine residues. The resulting ArMs proved to be effective at catalysing an enantioselective Friedel-Crafts reaction. SCP_Q111BpyAla achieved the best selectivity with enantioselectivity of 70% (S). Both enantiomers of the product were obtained using the same protein scaffold and interestingly, even with Bpy at the same residue but incorporated using a different attachment strategy changes the selectivity of the novel catalyst. X-ray crystal structures of the SCP_Q111CBpy and SCP_Q111BpyAla ArMs with Cu(II) ions bound were successfully solved and used to improve our understanding of the active site of these ArMs.
References 1. F. Schwizer et al., Chem. Rev. , 2018 , 118 , 142–231. 2. A. G. Jarvis et al., Angew. Chem. Int. Ed. , 2017 , 44 , 13784–13788. 3. I. Drienovská, A. Rioz-Martinez, A. Draksharapu and G. Roelfes, Chem. Sci. , 2015 , 6 , 770-776. 4. J. Xie, W. Liu and P.G. Schultz, Angew. Chem. Int. Ed. , 2007 , 46 , 9239 –9242.
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