Hydroxynitrile Lyases with α/β‐Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

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.

Increasing the Reaction Rate of Hydroxynitrile Lyase fromHevea brasiliensistoward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters

“…Finally, At HNL favors the ( R )-mandelonitrile in contrast to Hb HNL’s preference for the ( S )-enantiomer, so the substrate orientations likely differs significantly. [17] …”

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

“…Andexer et al 112 docked the substrate in the active site and proposed a possible mechanism for At EST5, Scheme 21. The key feature is binding the nitrile substituent in the oxyanion hole.…”

“…112 The reaction begins with proton abstraction from the hydroxyl group of mandelonitrile by His236. Next, elimination of cyanide forms the carbonyl group.…”

“…112 AtEST5 binds the substrate in an orientation that differs from both esterases (carbonyl is not in oxyanion hole) and from Hb HNL (nitrile is in oxyananion hole). X-ray structures have not confirmed this proposed binding.…”

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

“…The structures solved so far show similarity to a/b-hydrolases, oxidoreductases, carboxypeptidases or Zn 2+ -dependent alcohol dehydrogenases [2,7]. Interestingly, members of the a/b-hydrolase fold show both (R)-and (S)-selectivity [8,9]. Most HNLs have been discovered in plants, and several have been cloned successfully and expressed in Escherichia coli or yeasts such as Pichia pastoris [10,11].…”