Enzymes by design: chemogenetic assembly of transamination active sites containing lysine residues for covalent catalysis.
Artificial enzymes can be created by covalent conjugation of a catalytic active group to a protein scaffold. Here, two transamination catalysts were designed via computer modeling and assembled by chemically conjugating a pyridoxamine moiety within the large cavity of intestinal fatty acid binding protein. Each catalyst included a lysine residue, introduced via site-directed mutagenesis, that promotes catalysis by covalent interactions with the pyridoxamine group. Evidence for such interactions include the formation of a Schiff base with the pyridoxal form of the catalyst and a rate versus pH dependence that is bell shaped; both of these features are manifested in natural transaminases. The resulting constructs operate with high enantioselectivity (83-94% ee) and increase the rate of reaction as much as 4200-fold over the rate in the absence of the protein; this is a modest (12-fold) increase in catalytic efficiency (kcat/KM) compared to the conjugate lacking the lysine residue. Most importantly, these artificial aminotransferases are the first examples of designed bioconjugates capable of covalent catalysis, highlighting the potential of this chemogenetic approach.
Häring
D
D
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Distefano
M D
MD
D002352:Carrier Proteins / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D015195:Drug Design; D004798:Enzymes / Q000138:chemical synthesis* / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D009363:Neoplasm Proteins; D011733:Pyridoxamine / Q000737:chemistry; D013237:Stereoisomerism; D000637:Transaminases / Q000138:chemical synthesis / Q000378:metabolism
D002352:Carrier Proteins; D004798:Enzymes; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D011733:Pyridoxamine; D000637:Transaminases; D008239:Lysine