4ZJ1

Crystal Structure of p-acrylamido-phenylalanine modified TEM1 beta-lactamase from Escherichia coli : V216AcrF mutant

Summary for 4ZJ1

• Entry DOI: 10.2210/pdb4zj1/pdb
• Related: 4ZJ2 4ZJ3
• Descriptor: Beta-lactamase TEM, GLYCEROL, 2-(N-MORPHOLINO)-ETHANESULFONIC ACID, ... (5 entities in total)
• Functional Keywords: noncanonical amino acid, beta-lactamase, protein evolution, evolutionary advantage, hydrolase
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 33389.18

Authors

Xiao, H.,Nasertorabi, F.,Choi, S.,Han, G.W.,Reed, S.A.,Stevens, R.C.,Schultz, P.G. (deposition date: 2015-04-28, release date: 2015-05-20, Last modification date: 2023-11-15)

Primary citation

Xiao, H.,Nasertorabi, F.,Choi, S.H.,Han, G.W.,Reed, S.A.,Stevens, R.C.,Schultz, P.G.
Exploring the potential impact of an expanded genetic code on protein function.
Proc.Natl.Acad.Sci.USA, 112:6961-6966, 2015

PubMed Abstract: With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216-AcrF mutation leads to conformational changes in key active site residues-both in the free enzyme and upon formation of the acyl-enzyme intermediate-that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

PubMed: 26038548
DOI: 10.1073/pnas.1507741112

Experimental method

X-RAY DIFFRACTION (1.54 Å)