Inversion of stereoselectivity by applying mutants of the hydroxynitrile lyase from Manihot esculenta.
The influence of Trp128-substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) on the stereoselectivity of MeHNL-catalyzed HCN additions to aldehydes with stereogenic centers, which yield the corresponding cyanohydrins, is described. In rac-2-phenylpropionaldehyde (rac-1) reactions, wild-type (wtMeHNL) and all MeHNL Trp128 mutants are highly (S)-selective toward the (R) enantiomer of rac-1; this results exclusively in (2S,3R)-cyanohydrin ((2S,3R)-2) with > or =96 % de. The (S) enantiomer of rac-1, however, only reacts (S)-selectively with wtMeHNL to give (2S,3S)-2 with 80 % de, whereas with Trp128 mutants, (R) selectivity increases with decreasing size of the amino acids exchanged. The MeHNL W128A mutant is exclusively (R)-selective, resulting in (2R,3S)-2 with 86 % de. The reaction behavior of rac-phenylbutyraldehyde (rac-5) is comparable with rac-1, which also inverts the stereoselectivity from (S) to (R) when the enzyme is exchanged from wtMeHNL to the W128A mutant. Stereogenic centers not adjacent to the aldehyde group, as in 7 and 9, do not influence the stereoselectivity of MeHNL catalysis, and (S) selectivity is observed in all cases. Stereoselectivity and inversion of stereoselectivity of MeHNL Trp128 mutant-catalyzed cyanohydrin formation can be explained and rationalized with crystal-structure-based molecular modeling.
Bühler
Holger
H
Institut für Organische Chemie der Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
Miehlich
Burkhard
B
Effenberger
Franz
F
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D006856:Hydrogen Cyanide / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D002365:Manihot / Q000201:enzymology*; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D013237:Stereoisomerism; D013379:Substrate Specificity
D000447:Aldehydes; C421488:3-phenylbutanal; D006856:Hydrogen Cyanide; D000446:Aldehyde-Lyases; C087495:acetone-cyanohydrin lyase