Abstract
Hydroxynitrile lyases (HNL) are involved in the catabolism of cyanogenic glycosides in cyanogenic plants and are powerful tools in the stereoselective synthesis of cyanohydrins. The recent cloning of the hydroxynitrile lyase from flax ( Linum usitatissimum; LuHNL) reveals that this enzyme defines a novel class of HNL. Thorough biochemical and mutational analysis of LuHNL have been hampered by low expression levels of the recombinant enzyme in Escherichia coli. To overcome this impediment, we have cloned a myc-His-tagged LuHNL-cDNA under control of the methanol-inducible AOX1 (alcohol oxidase) promotor of Pichia pastoris and introduced it in the SMD1168 strain. Recombinant LuHNL was kinetically indistinguishable from the authentic flax enzyme. Immobilized LuHNL was used for synthesis of several aliphatic ( R)-cyanohydrins in a preparative scale to analyze the products according to enantiomeric excess and yield of reaction. LuHNL has significant homologies to members of the Zn 2+-containing alcohol dehydrogenases (Zn 2+-ADHs). In particular, residues responsible for coordination of Zn 2+ ions or fulfilling structural or functional tasks in Zn 2+-ADHs are conserved. We found about 2–4 mol zinc per mol of recombinant LuHNL using atom absorption spectroscopy in a non His-tagged version of LuHNL. Using site-directed mutagenesis, we substituted several of the conserved residues against alanine in LuHNL and found that in most cases, HNL-activity was impaired. Hence, it seems that LuHNL and Zn 2+-ADHs have similar structural requirements with respect to maintaining a catalytically active structure. Residues essentially involved in catalysis of Zn 2+-ADHs are also of functional importance in LuHNL, suggesting that the removal of the proton from alcohol and cleavage of cyanohydrins can be fulfilled by similar active site structures.
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