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

Abstract

The hydroxynitrile lyase from cassava (Manihot esculenta Crantz) (EC 4.1.2.37) catalyzes the decomposition of the achiral ␣-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 ␣-hydroxy acids, ␣-hydroxy ketones, or ␤-ethanolamines, all of which are important building blocks in organic synthesis. Inhibition of hydroxynitrile lyase from M. esculenta (MeHNL) by serine-and histidinemodifying 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 ␣/␤ 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 nucleophilehistidine-acid, suggesting that MeHNL belongs to the ␣/␤ hydrolase fold group of enzymes. In contrast to all other enzymes having a catalytical triad, HNLs catalyze no net hydrolytic reactions.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). In recent years these enzymes have received increasing attention due to their potential use as catalysts for the stereoselective synthesis of chiral cyanohydrins (2-6).To date, HNLs from various dicotyledons (7, 8) as well as from Sorghum bicolor (SbHNL) 1 (9, 10), a monocotyledone, and Phlebodium aureum (PhaHNL) (11), a fern, have been biochemically characterized. HNLs comprise a heterogenous group of enzymes with homo-and heteromers as the active enzyme, glycoproteins and nonglycoproteins, as well as flavoproteins. The molecular mass of HNL subunits ranges from 20 to 60 kDa (7-11). The heterogeneity of their properties suggests that most of these enzymes have independently evolved (convergent evolution). In fact, the lack of homologies among the amino acid sequences of the recently cloned HNLs from Prunus serotina (PsHNL) (12), Manihot esculenta (Me-HNL) (13), and S. bicolor (14) emphasize this idea. While PsHNL and MeHNL show none or only limited homology to other known proteins (12, 13), SbHNL has astonishingly high sequence homology with serine carboxypeptidases, especially with those from wheat. It appears of particular relevance that SbHNL shares the catalytical triad Ser, Asp, and His with these enzymes (14). The catalytical...

Cyanogenesis in Cassava1

“…As shown in Figure 1A, purified cassava HNL had a subunit molecular mass of 28.5 kD, similar to that reported by Hughes et al (1994) and Wajant and Pfizenmaier (1996). The native enzyme, however, had a molecular mass of 50.1 kD, suggesting that it was a dimer (Fig.…”

“…1B). These results are in contrast to those of Hughes et al (1994), Chueskul andChulavatnatol (1996), and Wajant and Pfizenmaier (1996). Hughes et al (1994) reported that the native molecular mass of HNL was a 92-kD homotrimer, whereas the latter two groups reported that the native enzyme was a tetramer of 102 to 110 kD, respectively.…”

“…tein h Ϫ1 ) and a lower K m (0.93 mm) than those previously reported for HNL isolated from whole leaves (Kakes and Harvoort, 1992;Wajant and Pfizenmaier, 1996). Furthermore, the enzyme exhibited typical Michaelis-Menten kinetics in marked contrast to the results reported by Hughes et al (1994).…”

“…Hydrolysis of linamarin yields an unstable hydroxynitrile intermediate, acetone cyanohydrin, plus Glc. Acetone cyanohydrin spontaneously decomposes to acetone and HCN at pH Ͼ5.0 or temperatures Ͼ35°C and can be broken down enzymatically by HNL (Cutler and Conn, 1981;Yemm and Poulton, 1986;Wajant and Mundry, 1993;Wajant et al, 1994;White et al, 1994;White and Sayre, 1995;Zheng and Poulton, 1995;Hasslacher et al, 1996;Wajant and Pfizenmaier, 1996).…”

The structure of a complex of the lactonohydrolase zearalenone hydrolase with the hydrolysis product of zearalenone at 1.60 Å resolution

“…The second hydrolysis mechanism is typically referred to as the general base mechanism. This typically begins with substrate deprotonation (Sun et al, 2014;Wajant & Pfizenmaier, 1996;Zuegg et al, 1999). In the case of ZHD, this enzyme-product structure provides the final state of catalysis.…”