Metal Catalytic Center And Directed Evolution Of The Chlorothalonil Hydrolytic Dehalogenase(Chd)

Dechlorinases encoded by microorganisms play a key role in the detoxification and dechlorination of chlorinated aromatic compounds.Great attention has been payed to these dechlorinases because of their potential applications in chemical industry and bioremediation.The mechanisms of dehalogenation of halogenated aromatic compounds include replacement by thiolytic,reduction,oxidation,and hydrolysis.There is only one dechlorinase system,the 4-chlorobenzate dehalogenase,which is capable of hydrolyzing the halogen atom from chlorinated aromatic compounds,was reported.However,the dechlorinase system needs three separate enzymes to cooperation,and needs CoA and ATP as cofacters.Chlorothalonil hydrolytic dehalogenase(Chd)reported in our laboratory is able to hydrolytically dechlorinate the chlorinated aromatic fungicide chlorothalonil(2,4,5,6-tetrachloroisophthalonitrile),which is the second most widely used agricultural fungicide in the United States with 5 million kilograms applied annually.Chd catalyzes a hydroxyl substitution at the 4-chlorine atom of chlorothalonil and is independent of cofactors such as coenzyme A(CoA)and ATP.Chd contains a putative conserved domain of the metallo-β-lactamase superfamily and has the highest identity with metallohydrolases.More ever,Chd was completely inhibited by the Zn2+-chelating metalloprotease inhibitor 1,10-phenanthroline(1 mM),and the catalytic activity was recovered by the subsequent supplementation of Zn2+,showing that Chd need to hold a tightly bound divalent cation essential for activity.Chd has a narrow substrate range and can only act on the 4-site chlorine atom of chlorothalonil.By the method of site-directed evolution,the catalytic activity and even the substrate range of Chd could be improved or expanded.Different divalent metal ions(Co2+,Cd2+,Mn2+,Cat+,Zn2+,Mg2+,Hg2+,Cu2+,Ni2+,Cr2+ and Fe2+)were used to see whether other metals could replace the metal center Zn2+ or not.The apo-Chd was first obtained by 1,10-phenanthroline chelation and subsequent dialysis.It was found that some other divalent cation such as cobalt(Co)and cadmium(Cd)could substitute the metal center zinc(Zn),and manganese(Mn)could replace Zn in some content.If the activity of wild type Zn2+-Chd was taken as 100%,the activity of mutanat Cot+-Chd was improved to 120%,while,the activities of Cd2+-Chd and Mn2+-Chd were decreased to 91.6%and 57%,respectively.The kinetic parameters of these metal-substituted Chd were also analyzed.It was found that the Km value of mutant Chd was also changed.Different metal replacement also changed the optimal temperature,pH value,thermal stability and pH stability of the mutant Chd.In this study,the metal catalytic center of Chd was systematically investigated.Inductively Coupled Plasma-Atomic Emission Spectrometry(ICP-AES)was first used to determine the metal content of Chd.Based on the protein concentration of Chd,it was calculated that there are 2.14 equivalents of zinc/mol of protein,showing that the active site of Chd contains a binuclear(Zn2+-Zn2+)center.In combination of site-directed mutagenesis,the residues that coordinate the metal center were also predicted by 3D LigandSite(http://www.sbg.bio.ic.ac.uk/3dligandsite/)and site-directed mutation was used to confirm that Hisl28,Asp130,Hisl31,Asn230,and His271 were the metal ligating amino acid residues.Error Prone-PCR and DNA shuffling was used to generate Chd variants and five mutants were obtained.One mutant,TB-1,which had two site mutations(L109H,S303G),displayed a 2.5-fold increase in specific activity.Another varant obtained by DNA shuffling displayed a 3.7-fold increase in specific activity.