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Rational Protein Design of ThDP‐Dependent Enzymes—Engineering Stereoselectivity
Author(s) -
Gocke Dörte,
Walter Lydia,
Gauchenova Ekaterina,
Kolter Geraldine,
Knoll Michael,
Berthold Catrine L.,
Schneider Gunter,
Pleiss Jürgen,
Müller Michael,
Pohl Martina
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700598
Subject(s) - stereoselectivity , chemistry , steric effects , stereochemistry , benzaldehyde , active site , acetaldehyde , pseudomonas putida , aldehyde , rational design , selectivity , enzyme , organic chemistry , catalysis , biology , ethanol , genetics
Benzoylformate decarboxylase (BFD) from Pseudomonas putida is an exceptional thiamin diphosphate‐dependent enzyme, as it catalyzes the formation of ( S )‐2‐hydroxy‐1‐phenylpropan‐1‐one from benzaldehyde and acetaldehyde. This is the only currently known S ‐selective reaction (92 % ee ) catalyzed by this otherwise R ‐selective class of enzymes. Here we describe the molecular basis of the introduction of S selectivity into ThDP‐dependent decarboxylases. By shaping the active site of BFD through the use of rational protein design, structural analysis, and molecular modeling, optimal steric stabilization of the acceptor aldehyde in a structural element called the S pocket was identified as the predominant interaction for adjusting stereoselectivity. Our studies revealed Leu461 as a hot spot for stereoselectivity in BFD. Exchange to alanine and glycine resulted in variants that catalyze the S ‐stereoselective addition of larger acceptor aldehydes, such as propanal with benzaldehyde and its derivatives—a reaction not catalyzed by the wild‐type enzyme. Crystal structure analysis of the variant BFDL461A supports the modeling studies.