Construction And Application Of Artificial Dicopper Metalloenzyme Base On Rubredoxin

Artificial metalloenzymes are a new class of enzyme molecules that introduce catalytically active metal cofactors into protein scaffolds.In the construction of artificial metalloenzymes,metal cofactors are mainly anchored in protein scaffolds in three ways: coordinative anchoring,supramolecular anchoring,and covalent anchoring.Coordination anchoring requires the coordination of ligand amino acids and protein structure to fix metal cofactors to protein scaffolds;Supramolecular anchoring requires the binding of specific proteins to their highaffinity metal cofactor-containing small molecules.Both methods have the limitation of narrow scope of application.Covalent anchoring uses chemical methods to modify amino acid side chains in protein scaffolds to introduce non-natural metal ligands to bind metal cofactors.This method has a wider range of applications and potential.However,when introducing unnatural ligands by this method,it is often not selective for multiple identical angisuan in the sequence,and it is difficult to obtain protein scaffolds modified at specific sites.Chemical protein synthesis is a relatively mature technology,which can introduce nonnatural metal ligands at specific amino acid sites in the process of Solid-Phase Peptide Synthesis(SPPS),and further combine various ligands through peptide ligation reactions.The assembly of peptide fragments into corresponding full-length proteins is expected to overcome the limitations of non-directional modification of covalent anchoring.Rubredoxin from extreme thermophilic archaea has a simple and stable structure and is suitable as a model protein for exploring chemical total synthesis to construct artificial metalloenzymes.This protein is the smallest known iron-sulfur cluster protein,containing 54 amino acid residues.There have been many reports to replace the iron center of the protein with other metals such as copper,cobalt,and nickel,but there are few works on further transformation into artificial metalloenzymes.In this paper,a method combining chemical protein synthesis and covalent anchoring was developed to construct a metalloprotein containing dicopper centers using Rubredoxin protein scaffolds.To obtain this dicopper protein,the acquisition strategy of wild-type Rubedoxin was first explored and the synthetic route from the C-terminal to the N-terminal was determined.Secondly,the artificial metalloenzyme fragment of Rubredoxin was obtained by SPPS,and Dipicolylamine(Dpa)ligand was introduced by covalent anchoring.Subsequently,the fulllength target protein was obtained by Native Chemical Ligation(NCL),copper ions were introduced,and its spectroscopic properties were characterized.Finally,the catalytic properties of the enzyme in phenol oxidation and monooxygenation reactions were tested.Although the catalytic efficiency of this enzyme in the monooxygenation catalysis experiment is not high,the work still lays a technical foundation for the use of iron-sulfur protein family proteins such as Rubedoxin as artificial metalloenzyme scaffolds in the future,and also for the synthesis of artificial metalloproteinases.Broadening provides guidance...