Genome Mining And Molecular Engineering Of Nitrile Reductase From Pectobacterium Carotovorum

Nitrile reductase catalyzes the reduction of 7-cyano-7-deazaguanine(PreQ0)to 7-aminomethyl-7-deazaguanine(PreQ1)only found in bacteria,which is different from traditional nitrile-converting enzymes including nitrilases,nitrile hydratases,nitrogenases,and oxygenases,the discovery of this novel nitrile-converting enzyme would pave the way for developing environmentally sustainable nitrile-reducing biocatalysts and represent an important alternative to metal catalysts.However,little progress has been made towards the application of this biocatalysis reaction for organic synthesis since the initial discovery of nitrile reductase for over ten years.One of the potential reasons may be due to the poor evolvability of the T-fold enzymes and the kinetically sluggish reaction catalyzed by QueF.Searching for new nitrile reductases or engineering the existent nitrile reductase may be a promising alternative to develop synthetic useful nitrile reductase biocatalyst.To explore more nitrile reductases with different characteristics(e.g.specific activity,thermostability,etc)for potential synthetic applications,a new nitrile reductase,designated as PcNR,was discovered from the genome of Pectobacterium carotovorum,with a moderate specific activity of 270 U/g protein towards its natural substrate,PreQ0.The PcNR was subsequently overexpressed in E.coli BL21(DE3),purified to homogeneity and then its biochemical properties were examined.The optimical reaction temperature and pH were determined to be 40? and pH 7.5,respectively.The thermal deactivation process of PcNR obeys first-order kinetics,with half-lives of 73.7 h,5.3 h and 10 min measured at 30,40,and 50?,respectively.The kinetic constants were also determined with respect to both of the substrates NADPH and PreQ0,giving kcat of 0.35 s-1 and 0.20 s-1,and KM of 2.7 ?M and 3.4?M.To shed some light on the structural information of PcNR and figure out the relationship between structure and function,a homology model was built by the Swiss Model on-line tool(http://swissmodel.expasy.org)using crystal structure of VcNR(PDB ID:3UXV.1.A)as a template and validated by PROCHEK,which was helpful for further engineering of this novel but less-elucidated enzyme.By structure-guided site-directed mutagenesis and saturation mutagenesis,we were able to increase the catalytic efficiency of PcNR towards PreQ0 by about 2-fold.Structural analysis show that the mutation might alter the flexibility of the loop and change the microenvironment of the catalytic cysteine and the enhanced activity is accompanied by the removal of solvent-exposed hydrophobic group.