Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot.
Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild-type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism-inspired and structure-guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot.
Abt. Molekulare Enzymologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
Cindy
C
Wechsler
Abt. Molekulare Enzymologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
Danilo
D
Meyer
Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg im Breisgau, Germany.
Sabrina
S
Loschonsky
Abt. Molekulare Enzymologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
Lisa-Marie
LM
Funk
Abt. Molekulare Strukturbiologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
Piotr
P
Neumann
Abt. Molekulare Strukturbiologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
Ralf
R
Ficner
Abt. Molekulare Enzymologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
Florian
F
Brodhun
Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg im Breisgau, Germany.
Michael
M
Müller
Abt. Molekulare Enzymologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany. ktittma@gwdg.de.
Kai
K
Tittmann
D000096:Acetone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D005233:Fatty Alcohols / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D017355:Zymomonas / Q000201:enzymology
D005233:Fatty Alcohols; D000096:Acetone; C011159:acyloin; C035293:1-hydroxy-1-phenyl-2-propanone; D011767:Pyruvate Decarboxylase
asymmetric catalysis; biocatalysis; cofactors; enantioselectivity; enzyme catalysis