Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis.
The hydroxynitrile lyase from cassava (Manihot esculenta Crantz) (EC 4.1.2.37) catalyzes the decomposition of the achiral alpha-hydroxynitrile acetone cyanohydrin into HCN and acetone during cyanogenesis of damaged plants. This enzyme can also be used for stereoselective synthesis of a wide array of (S)-cyanohydrins by addition of HCN to aldehydes or ketones. Optically active cyanohydrins are interesting intermediates for the synthesis of alpha-hydroxy acids, alpha-hydroxy ketones, or beta-ethanolamines, all of which are important building blocks in organic synthesis. Inhibition of hydroxynitrile lyase from M. esculenta (MeHNL) by serine- and histidine-modifying reagents suggests involvement of active site seryl and histidyl residues. Furthermore, serine 80 of MeHNL is part of the active site motif Gly-X-Ser-X-Gly/Ala, often considered as the hallmark of catalytic triads having independently evolved in four groups of enzymes: the alpha/beta hydrolase fold enzymes, subtilisins, the cysteine proteases, and the eukaryotic serine proteases. By site-directed mutagenesis, three residues critical for enzyme activity have been identified: serine 80, aspartic acid 208, and histidine 236. These residues may be directly involved in MeHNL-catalyzed decomposition of cyanohydrins, providing evidence for a catalytical triad in HNLs, too. The order of the catalytic triad residues in the primary sequence of MeHNL is nucleophile-histidine-acid, suggesting that MeHNL belongs to the alpha/beta hydrolase fold group of enzymes. In contrast to all other enzymes having a catalytical triad, HNLs catalyze no net hydrolytic reactions.
Wajant
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Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Pfizenmaier
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D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D002365:Manihot; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009570:Nitriles / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism
D009570:Nitriles; D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; C010833:acetone cyanohydrin; D000446:Aldehyde-Lyases; C087495:acetone-cyanohydrin lyase