Identification of Potential Active-site Residues in the Hydroxynitrile Lyase from Manihot esculenta by Site-directed Mutagenesis

Harald Wajant,Klaus Pfizenmaier

doi:10.1074/jbc.271.42.25830

Harald Wajant, Klaus Pfizenmaier

Open Access

https://doi.org/10.1074/jbc.271.42.25830

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Abstract

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.

Highlights

Hydroxynitrile lyases, which catalyze the dissociation of ␣-hydroxynitriles into carbonyle and HCN (Fig. 1), are described for several plant species of widely differing phylogenetic origin [1]
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 ␣/␤ hydrolase fold enzymes, subtilisins, the cysteine proteases, and the eukaryotic serine proteases
Site-directed mutagenesis further shows that histidine 236 and aspartic acid 208 are important for enzyme activity

Summary

Introduction

Hydroxynitrile lyases, which catalyze the dissociation of ␣-hydroxynitriles (cyanohydrins) into carbonyle and HCN (Fig. 1), are described for several plant species of widely differing phylogenetic origin [1]. 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 ␣/␤ hydrolase fold enzymes, subtilisins, the cysteine proteases, and the eukaryotic serine proteases.

Results

Conclusion