Abstract
Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.
Highlights
Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins into the corresponding carbonyl compound and hydrocyanic acid (Scheme 1)
The necessity of a general base in HNL catalysis has been proposed more than 45 years ago by Becker and Pfeil,[13] who predicted the requirement for a positive charge in the active sites of HNLs to stabilize the negative charge evolving at the cyano group during the reaction
We report the crystal structure at 2.5 resolution (Table 1) of the HNL from A. thaliana, for which sequence similarities and mutational data for putative active-site residues[14] had sug
Summary
Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins into the corresponding carbonyl compound and hydrocyanic acid (Scheme 1). Structure and Mechanism of AtHNL (root-mean-square deviations (RMSD) of 0.3 for a superposition of 230 out of 256 Ca-atoms) and biochemical properties that we used the (somewhat better characterized) HbHNL as prototype for (S)-HNLs with an a/b-hydrolase fold Their enzymatic mechanism involves the Ser-His-Asp catalytic triad as a general base deprotonating the cyanohydrin substrate with the histidine residue being the actual base and the serine hydroxyl group acting as a mediator.[12] The necessity of a general base in HNL catalysis has been proposed more than 45 years ago by Becker and Pfeil,[13] who predicted the requirement for a positive charge in the active sites of HNLs to stabilize the negative charge evolving at the cyano group during the reaction. What is the reaction mechanism of AtHNL, how is the negative charge of the nascent cyanide ion stabilized?
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