Construction Of Protein Catenane On Bio-catalyst And Study On Hydroxylation Of Canucin Lasso Peptide

In recent years,the topological engineering of protein and the synthesis mechanism of topological proteins have attracted wide attention.This article studies these two issues separately.The application of protein topology engineering shows good results in changing protein stability and function.Inspired by the art of nature where backbone cyclization is frequently adopted to enhance the stability of peptide natural products and non-thermostable enzymes.Herein,based on the concept of topological structure modification,we reported protein topology engineering of an industrial thermolabile gamma lactamase via catenation.Two different protein catenanes was successfully constructed via SpyTag/SpyCatcher modules and two different peptide dimer domains.These two designed protein catenanes were functionally biosynthesized in vivo by Escherichia coli.Comparation the biochemical properties revealed that protein topology plays a key roles in the stablility of gamma lactamase.Protein catenation could enhance both the thermostability and proteolytic stability of the target enzymes.Moreover,the Catl-MhIHL-V 54L and Cat2-MhIHL-V54L displayed a 3.2-fold and 3.8-fold higher enzyme efficiency(Kcat/Km)compared to the wild-type enzyme.Therefore,our studies proved that protein catenanes construction could be developed as a general strategy to strengthen industrial biocatalysts by unique mechanisms,distinct from those possible through conventional mutagenesis schemes and might have a wide application in fine chemical industry.In addition to catenane forms,topoproteins also have lasso peptides.Lasso peptides belong to ribosomal synthesis and post-translational modified peptides(RiPPs),which have a unique lasso knot topology.Recently,by activating the silent biosynthetic gene cluster(BGCs)in Streptomyces canus NRRL B3980,the post-translational hydroxylated lasso peptide canucin A was discovered.Therefore,?-OH-Asp modification at the C-terminal of canucin A is the second subject of this paper.Through biochemical experiments,we demonstrated that the Fe/20G-dependent enzyme CanE installs a hydroxyl group on the precursor peptide CanA in vitro.The results of the enzymatic properties of CanE showed that the optimal catalytic temperature was 30?,and the relative activity was above 92%under the reaction conditions of pH 5-8.Under the optimal reaction conditions,the Km was 0.24±0.02 mM,and the Kcat was 0.02± 2.17*10-5 min-1.