Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with alpha/beta-hydrolase fold.
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.
Institute of Molecular Enzyme Technology, Heinrich-Heine University Duesseldorf, Juelich Forschungszentrum, Juelich, Germany.
Jan-Karl
JK
Guterl
Jennifer N
JN
Andexer
Torsten
T
Sehl
Jan
J
von Langermann
Ilona
I
Frindi-Wosch
Tobias
T
Rosenkranz
Jörg
J
Fitter
Karl
K
Gruber
Udo
U
Kragl
Thorsten
T
Eggert
Martina
M
Pohl
D000097:Acetonitriles / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D028482:Hevea / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics*; D002365:Manihot / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D000097:Acetonitriles; D010940:Plant Proteins; D011994:Recombinant Proteins; C045516:mandelonitrile; D006867:Hydrolases; D000446:Aldehyde-Lyases