Publications

• Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat).

  Ueatrongchit T., Kayo A., Komeda H., Asano Y., H-Kittikun A.

  A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which sug ... >> More

  A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K(m) of 161 microM and a k(cat)/K(m) of 348 s(-1) mM(-1) for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 degrees C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO(4), HgCl(2), AgNO(3), FeCl(3), beta-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k(cat)/K(m)) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one. << Less

  Biosci Biotechnol Biochem 72:1513-1522(2008) [PubMed] [EuropePMC]

• Studies on the kinetics of cyanohydrin synthesis and cleavage by the the flavoenzyme oxynitrilase.

  Jorns M.S.

  Almond oxynitrilase (D-alpha-hydroxynitrile lyase, EC 4.1.2.10) catalyzes the reversible condensation of HCN with aldehydes to form D-alpha-hydroxynitriles. Steady-state kinetic parameters for cleavage and synthesis of mandelonitrile and vanillin cyanohydrin were determined at pH 5.5 which is near ... >> More

  Almond oxynitrilase (D-alpha-hydroxynitrile lyase, EC 4.1.2.10) catalyzes the reversible condensation of HCN with aldehydes to form D-alpha-hydroxynitriles. Steady-state kinetic parameters for cleavage and synthesis of mandelonitrile and vanillin cyanohydrin were determined at pH 5.5 which is near the pH optimum of the enzyme. Benzaldehyde and vanillin act as competitive inhibitors of cyanohydrin cleavage while noncompetitive inhibition was observed for HCN. The results are consistent with an ordered uni bi mechanism in which aldehyde is the first substrate bound. Competitive inhibition of cyanohydrin cleavage was observed with various carboxylic acids, alcohols and inorganic anions. The effect of structure on the binding of these inhibitors suggests that the active site of oxynitrilase is located near a hydrophobic region and a positively charged group. Inhibitors which are reasonable analogues for cyanide anion, such as azide and thiocyanate, do not bind to the enzyme-aldehyde complex. This suggests that during cyanohydrin formation the species which binds to the enzyme-aldehyde complex is HCN rather than CN-. << Less

  Biochim. Biophys. Acta 613:203-209(1980) [PubMed] [EuropePMC]

• An R-selective hydroxynitrile lyase from Arabidopsis thaliana with an alpha/beta-hydrolase fold.

  Andexer J., von Langermann J., Mell A., Bocola M., Kragl U., Eggert T., Pohl M.

  Angew. Chem. Int. Ed. Engl. 46:8679-8681(2007) [PubMed] [EuropePMC]

• Substrate binding in the FAD-dependent hydroxynitrile lyase from almond provides insight into the mechanism of cyanohydrin formation and explains the absence of dehydrogenation activity.

  Dreveny I., Andryushkova A.S., Glieder A., Gruber K., Kratky C.

  In a large number of plant species hydroxynitrile lyases catalyze the decomposition of cyanohydrins in order to generate hydrogen cyanide upon tissue damage. Hydrogen cyanide serves as a deterrent against herbivores and fungi. In vitro hydroxynitrile lyases are proficient biocatalysts for the ster ... >> More

  In a large number of plant species hydroxynitrile lyases catalyze the decomposition of cyanohydrins in order to generate hydrogen cyanide upon tissue damage. Hydrogen cyanide serves as a deterrent against herbivores and fungi. In vitro hydroxynitrile lyases are proficient biocatalysts for the stereospecific synthesis of cyanohydrins. Curiously, hydroxynitrile lyases from different species are completely unrelated in structure and substrate specificity despite catalyzing the same reaction. The hydroxynitrile lyase from almond shows close resemblance to flavoproteins of the glucose-methanol-choline oxidoreductase family. We report here 3D structural data of this lyase with the reaction product benzaldehyde bound within the active site, which allow unambiguous assignment of the location of substrate binding. Based on the binding geometry, a reaction mechanism is proposed that involves one of the two conserved active site histidine residues acting as a general base abstracting the proton from the cyanohydrin hydroxyl group. Site-directed mutagenesis shows that both active site histidines are required for the reaction to occur. There is no evidence that the flavin cofactor directly participates in the reaction. Comparison with other hydroxynitrile lyases reveals a large diversity of active site architectures, which, however, share the common features of a general active site base and a nearby patch with positive electrostatic potential. On the basis of the difference in substrate binding geometry between the FAD-dependent HNL from almond and the related oxidases, we can rationalize why the HNL does not act as an oxidase. << Less

  Biochemistry 48:3370-3377(2009) [PubMed] [EuropePMC]

• Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with alpha/beta-hydrolase fold.

  Guterl J.K., Andexer J.N., Sehl T., von Langermann J., Frindi-Wosch I., Rosenkranz T., Fitter J., Gruber K., Kragl U., Eggert T., Pohl M.

  Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concer ... >> More

  Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concerning their properties relevant for technical applications. The results are compared to available data of the structurally related S-HNL from Hevea brasiliensis (HbHNL), which is frequently applied in technical processes. Whereas substrate ranges are highly similar for all three enzymes, the stability of MeHNL with respect to higher temperature and low pH-values is superior to the other HNLs with alpha/beta-hydrolase fold. This enhanced stability is supposed to be due to the ability of MeHNL to form tetramers in solution, while HbHNL and AtHNL are dimers. The different inactivation pathways, deduced by means of circular dichroism, tryptophan fluorescence and static light scattering further support these results. Our data suggest different possibilities to stabilize MeHNL and AtHNL for technical applications: whereas the application of crude cell extracts is appropriate for MeHNL, AtHNL is stabilized by addition of polyols. In addition, the molecular reason for the inhibition of MeHNL and HbHNL by acetate could be elucidated, whereas no such inhibition was observed with AtHNL. << Less

  J. Biotechnol. 141:166-173(2009) [PubMed] [EuropePMC]

• A rapid and sensitive spectrophotometric assay for prunasin hydrolase activity employing purified mandelonitrile lyase.

  Gross M., Jacobs G.H., Poulton J.E.

  Anal Biochem 119:25-30(1982) [PubMed] [EuropePMC]