Mandelonitrile lyase from Ximenia americana L.: stereospecificity and lack of flavin prosthetic group.

Abstract

A mandelonitrile lyase (EC 4.1.2.10) that catalyzes the dissociation of (S)-(-)-mandelonitrile to benzaldehyde and hydrogen cyanide has been purified to apparent homogeneity from leaves ofXimenia americana L. (Olacaceae). The lyase was purified 122-fold with 38% yield by chromatography on carboxymethyl-celiulose and chromatofocusing. The enzyme had a pH optimum of 5.5, with a K. value of 280 ,uM.Activity toward 4-hydroxy-(R,S)-mandelonitrile was 77% of that observed with the endogenous substrate; no activity was observed toward the aliphatic substrate acetone cyanohydrin. The enzyme was stable at 40C and at room temperature for at least 1 month. Native and subunit molecular weights of 38,000 and 36,500, respectively, suggest the enzyme is a monomer. The isoelectric point was pH 3.9 as determined by isoelectric focusing. Staining with periodic acid-Schiff and fluoresceinlabeled concanavalin A reagents indicate this enzyme is a glycoprotein. In contrast to (R)-mandelonitrile lyases isolated from Prunus species, the Ximenia lyase does not appear to be a flavoprotein. A second enzyme that eluted from the chromatofocusing column at pH 4.0 was also active toward mandelonitrile. However, this form accounted for less than 10% of the total activity, and its specific activity was only 6% of that of the major component. Additional physical and kinetic studies suggested this activity may be due to a nonspecific enzyme that is active toward mandelonitrile.(R)-(+)-Mandelonitrile, produced by hydrolysis of cyanogenic glucosides that occur in members of the family Rosaceae, is reversibly dissociated into benzaldehyde and HCN by the enzyme mandelonitrile lyase, an a-hydroxynitrile Iyase (EC 4.1.2.10) (1). Pfeil and associates (2-4) reported that these enzymes, when isolated from the genus Prunus, were flavoproteins and speculated on the role of the flavin cofactor in a reaction not involving oxidation/reduction.Other workers (5-8) have examined the role of the flavin moiety and this has been the subject of considerable debate.Although the (R)-(+)-mandelonitrile lyases of rosaceous species appear to be flavoproteins (1), the analogous enzyme in Sorghum bicolor that utilizes 4-hydroxy-(S)-mandelonitrile as a substrate does not contain a flavin cofactor (9).Moreover, lyases in cassava (10) and flax (11), which utilize the aliphatic substrate acetone cyanohydrin, do not appear to be flavoproteins. Thus the (R)-(+)-mandelonitrile lyase of rosaceous species is atypical in its requirement for a flavin cofactor.The occurrence of the glucoside of (S)-(-)-mandelonitrile in Ximenia americana (family Olacaceae) (12) provided an opportunity to examine whether the lyase that acts on the enantiomer of (R)-(+)-mandelonitrile is a flavoprotein. Accordingly the a-hydroxynitrile Iyase of X. americana has been purified to apparent electrophoretic homogeneity and its properties have been examined. MATERIALS AND METHODS Materials. Leaves of X. americana were collected at Fort Desoto Park (Saint Petersburg, FL) and shipped to Davis, California...

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

“…First, mandelonitrile lyase (EC 4.1.2.10) catalyzes the natural substrate (R)-or (S)-mandelonitrile and is found in X. americana (S stereo selective), 3) Pr. subfamily (R stereo selective), 11,16) E. japonica (R), 22) and M. americana (R).…”

“…Depending on their stereo preferences for asymmetric cyanohydrin synthesis, HNLs are often divided into two classes: (S)-HNL and (R)-HNL. (S)-HNLs are found and investigated in Ximenia americana (XaHNL), 3) Manihot esculenta (MeHNL), 4) Hevea brasiliensis (HbHNL), 5) and Sorghum bicolor (SbHNL). 6) (R)-HNLs are Phlebodium aureum (PhaHNL), 7) Linum usitatissimum (LuHNL), 8,9) Arabidopsis thaliana (AtHNL), 10) and Pr.…”

Three‐dimensional structures of enzyme‐substrate complexes of the hydroxynitrile lyase from hevea brasiliensis

“…FAD-dependent HNLs accept~R!~ϩ!mandelonitrile as their biological substrate and appear to have evolved from FAD-dependent oxidoreductases Kuroki & Conn, 1989!. FAD-independent hydroxynitrile lyases accept a variety of~R!~Linacaeae; Xu et al, 1988!…”

“…FAD-independent hydroxynitrile lyases accept a variety of~R!~Linacaeae; Xu et al, 1988! and~S!~Eu-phoribiacae;Hughes et al, 1994;Olacaeae;Kuroki & Conn, 1989;Graminae;Wajant & Mundry, 1993! configured cyanohydrins as substrates.…”

Crystallographic studies and preliminary X-ray investigation of (S)-p-hydroxy-mandelonitrile lyase fromSorghum bicolor(L.)

“…HNL's of group I contain a flavineadenine dinucleotide (FAD) cofactor, are typically singlechain glycoproteins showing remarkable serological crossreactivity (Gerstner & Pfeil, 1972) and catalyze exclusi~,ely the decomposition of R-cyanohydrines. The second group of lyases is FAD-cofactor independent and forms a more heterogeneous group of enzymes, distinct in molecular mass, subunit composition, glycoprotein nature, substrate specificity and stereoselectivity of the enzymatic reaction (Xu, Singh &Conn, 1988;Kuroki & Conn, 1989;Hughes, de Carvalho & Hughes, 1994;. It has been suggested that this group of enzymes has originated by convergent evolution from different ancestral enzymes.…”