Several structurally different types of HNLs occur in nature, which likely originated by convergent evolution from different ancestral proteins.38 The enzyme from almond (PaHNL) was first crystallized in 1994 and the structure was solved by multiple wavelength anomalous dispersion of a mercury derivative.39 The first 3D structure analysis of PaHNL was performed in 2001.40 (R)-PaHNL from almond uses FAD as cofactor and is related to oxidoreductases; it exhibits HNL activity only in the oxidized form of FAD.41
The crystal structure of the HNL isolated from S. bicolor (SbHNL) was determined in a complex with the inhibitor benzoic acid.42 The folding pattern of SbHNL is similar to that of wheat serine carboxypeptidase (CP-WII)43a and alcohol dehydrogenase.4313 A unique two-amino acid deletion in SbHNL, however, is forcing the putative active site residues away from the hydrolase binding site toward a small hydrophobic cleft, thereby defining a completely different active site architecture where the triad of a carboxypeptidase is missing.
The most extensively investigated HNL structures are those from H. brasiliensis (HbHNL)44 and M. esculenta (MeHNL),45 which are highly homologous (76% identity). For MeHNL, the crystal structure of the wild-type enzyme complexed with acetone has been reported in 2001 (Fig. 1).45
Both enzymes belong to the family of a,(-hydrolases.43 The active site of MeHNL is located inside the protein and connected to the outside through a small channel, which is covered by the bulky amino acid tryptophane 128.45 It was possible to obtain the crystal structure of the complex with the natural substrate acetone cyanohydrin with the mutant Ser80A1a of MeHNL. This complex allowed the determination of the mode of substrate binding in the active site.45 A summary of 3D structures of known HNLs was published recently.4647
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