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Hydroxynitrile lyases(HNLs)catalyze the conversion of chiral cyanohydrins to hydrocyanic acid(HCN)and aldehyde or ketone.Hydroxynitrile lyase from Arabidopsis thaliana(AtHNL)is the first R-selective HNL enzyme containing an α/β-hydrolases fold.The catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012.Two computational models were constructed,and two possible reaction pathways were considered.In path A,the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly,and then the cleavage of C1-C2 bond and the rotation of generated cyanide ion(CN-)anion follow.The C1-C2 bond cleavage and CN-rotation correspond to very similar energy barriers(13.1 and 13.4 kcal/mol),and both of them are rate limiting.In path B,the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1-C2 bond take place in concerted manner,which corresponds to the highest energy barrier of 17.1 kcal/mol.In both of the pathways,the catalytic triad(His236,Ser81 and Asp208)acts as the general acid/base,and the geneated CN-is stabilized by the hydroxyl group of Ser81 and the main-chain NH-groups of Ala13 and Phe82.