title: Rational protein design of ThDP-dependent enzymes-engineering stereoselectivity.
abstract: 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.
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine University Düsseldorf, 52426 Jülich, Germany.
authors.forename: Dörte
authors.initials: D
authors.lastname: Gocke
authors.forename: Lydia
authors.initials: L
authors.lastname: Walter
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Gauchenova
authors.forename: Geraldine
authors.initials: G
authors.lastname: Kolter
authors.forename: Michael
authors.initials: M
authors.lastname: Knoll
authors.forename: Catrine L
authors.initials: CL
authors.lastname: Berthold
authors.forename: Gunter
authors.initials: G
authors.lastname: Schneider
authors.forename: Jürgen
authors.initials: J
authors.lastname: Pleiss
authors.forename: Michael
authors.initials: M
authors.lastname: Müller
authors.forename: Martina
authors.initials: M
authors.lastname: Pohl
mesh_terms: D015195:Drug Design; D004798:Enzymes / Q000737:chemistry*; D015202:Protein Engineering; D013237:Stereoisomerism; D013835:Thiamine Pyrophosphate / Q000737:chemistry*
chemical_list: D004798:Enzymes; D013835:Thiamine Pyrophosphate