Reaction mechanism of cyanogenesis

Mechanisms of cyanogenesis have been published first based on the crystal structures of SbHNL from S. bicolor42 and of PaHNL from almonds.47 In this article, the mechanism of cyanogenesis will be discussed on the most proven mechanism of MeHNL from M. esculenta.45

Combining X-ray structure and site-directed mutagenesis results in a mechanism as shown in Scheme 10 in which His236 acts as a general base abstracting a proton from Ser80, thereby allowing proton transfer of the cyanohydrin to Ser80. The His236 imidazolium cation then facilitates leaving of the cyano group by proton transfer (Scheme 10).45 It was demonstrated that the active site lysine residue Lys237 in MeHNL is necessary for the catalytic activity.48

The (S)-enantioselectivity of MeHNL can be explained considering Fig. 1. As shown in the figure, the carbonyl oxygen is hydrogen bonded to Ser80 and Thr11. One methyl group of acetone (C1) is held in position by van der Waals contacts to Leu149, Thr11 and Ile12. The side chains of these residues define a

CYS 81

CYS 81

TRP 128

Figure 1: Structure of selected active site residues of MeHNL complexed with acetone.

TRP 128

Figure 1: Structure of selected active site residues of MeHNL complexed with acetone.

small hydrophobic site S1. The second methyl group (C3) points in the opposite direction toward the active site channel, defining the putative second subsite S2 in the binding cavity. Aldehydes are now fixed in a way that the small substituent hydrogen is situated in S1 and the larger substituent R in S2. This mode of binding suggests that the incoming cyanide exclusively attacks the Si-face of the carbonyl compound. This implies that HCN is deprotonated by His236, which is located on the Si-face of the substrate in order to be consistent with the (S)-enantioselectivity of the addition.45

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