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