Substrate binding in the FAD-dependent hydroxynitrile lyase from almond provides insight into the mechanism of cyanohydrin formation and explains the absence of dehydrogenation activity

Abstract

In a large number of plant species hydroxynitrile lyases catalyze the decomposition of cyanohydrins in order to generate hydrogen cyanide upon tissue damage. Hydrogen cyanide serves as a deterrent against herbivores and fungi. In vitro hydroxynitrile lyases are proficient biocatalysts for the stereospecific synthesis of cyanohydrins. Curiously, hydroxynitrile lyases from different species are completely unrelated in structure and substrate specificity despite catalyzing the same reaction. The hydroxynitrile lyase from almond shows close resemblance to flavoproteins of the glucose-methanol-choline oxidoreductase family. We report here 3D structural data of this lyase with the reaction product benzaldehyde bound within the active site, which allow unambiguous assignment of the location of substrate binding. Based on the binding geometry, a reaction mechanism is proposed that involves one of the two conserved active site histidine residues acting as a general base abstracting the proton from the cyanohydrin hydroxyl group. Site-directed mutagenesis shows that both active site histidines are required for the reaction to occur. There is no evidence that the flavin cofactor directly participates in the reaction. Comparison with other hydroxynitrile lyases reveals a large diversity of active site architectures, which, however, share the common features of a general active site base and a nearby patch with positive electrostatic potential. On the basis of the difference in substrate binding geometry between the FAD-dependent HNL from almond and the related oxidases, we can rationalize why the HNL does not act as an oxidase.

Figure 1

(A) Ligand binding sites in PaHNL1. Ribbon diagram of PaHNL1 (blue) with the flavin cofactor depicted in orange ball-and-stick representation indicating the substrate binding sites. Ligands are highlighted in red ball-and-stick representation, and the binding sites are numbered as 1 for the active site and 2 for the hydrophobic pocket. For clarity, carbohydrates at the glycosylation sites have been omitted in the figure. (B) Stereo representation of the active site region. The binding mode of the reaction product benzaldehyde with the active site water (red) and the modeled (R)-mandelonitrile (pink) are shown. Plausible hydrogen-bonding interactions are indicated by red dashed lines. The hydrogen-bonding network surrounding the active site residues His459 and His497 is indicated by black dashed lines.

Figure 2

(A) Water molecules within the active site of the native structure. Ribbon representation of the PaHNL1 active site region (blue) with important residues in ball-and-stick representation. F_o − F_c electron densities (gray) of active site water molecules in the native monoclinic crystal form. Plausible hydrogen-bonding interactions are indicated by dashed lines in cyan. (B) Benzaldehyde within the active site in the liganded structure. F_o − F_c density in the active site of the liganded structure (gray). Benzaldehyde and the active site water are depicted in red. The orientation of the active site region is shown as in the native structure. (C) Mandelonitrile binding site in a hydrophobic pocket. The F_o − F_c electron density of uncleaved mandelonitrile (in red ball-and-stick representation) in the hydrophobic pocket of the liganded structure is shown, indicating that this site has no catalytic function.

Figure 3

Stereo representation of the superposition of GMC oxidoreductase structures in complex with the respective substrate, inhibitor, or product with the PaHNL1 benzaldehyde complex structure and modeled (R)-mandelonitrile. Structures of CHOX with the steroid substrate dehydroisoandrosterone (25) (ligand shown in green), CBDH in complex with the inhibitor cellobiono-1,5-lactam (26) (ligand shown in dark blue), and POX in complex with the reaction product 2-keto-β-d-glucose (27) (ligand shown in light blue) are superimposed with PaHNL1−benzaldehyde and modeled (R)-mandelonitrile (ligands both shown in red). Superimposed FAD cofactors are depicted in orange, and the two important catalytic residues (His459 and His497 in PaHNL) are shown in light gray. Note the culmination of these structures in a single point close to the flavin isoalloxazine ring N5 atom (drawn in a close-up and highlighted by an arrow) and the mostly conserved active site residues.

Scheme 1. Proposed Mechanism of the PaHNL1 Cyanohydrin Cleavage Reaction

The reaction proceeds via general acid/base catalysis through residue His497. The cyanide is stabilized by the overall positive electrostatic potential in the active site region (indicated by a positive charge). The mechanistic details of the reprotonation of cyanide are currently unresolved, but it is likely that His459 is involved in this step. Plausible hydrogen-bonding interactions are indicated by dashed lines. Active site residues are labeled. According to the ordered Uni Bi mechanism benzaldehyde is the last product to leave the active site.