5VHT

E. coli chorismate mutase with orthogonal interface containing p-benzoyl phenylalanine

Summary for 5VHT

• Entry DOI: 10.2210/pdb5vht/pdb
• Descriptor: Chorismate Mutase (2 entities in total)
• Functional Keywords: p-benzoyl phenylalanine, orthogonal interface, chorismate mutase, mutagenesis, isomerase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 23272.83

Authors

Koh, M.,Nasertorabi, F.,Han, G.W.,Stevens, R.C.,Shultz, P.G. (deposition date: 2017-04-13, release date: 2017-05-10, Last modification date: 2023-11-15)

Primary citation

Koh, M.,Nasertorabi, F.,Han, G.W.,Stevens, R.C.,Schultz, P.G.
Generation of an Orthogonal Protein-Protein Interface with a Noncanonical Amino Acid.
J. Am. Chem. Soc., 139:5728-5731, 2017

PubMed Abstract: We have engineered the protein interface of the Escherichia coli chorismate mutase (EcCM) homodimer to be dependent on incorporation of a noncanonical amino acid (ncAA) at residue 72. The large hydrophobic amino acid p-benzoyl phenylalanine (pBzF) was substituted for Tyr72, which led to a catalytically inactive protein. A library of five residues (Leu25', Arg29', Leu76, Ile80' and Asp83') surrounding pBzF72 was generated and subjected to a growth based selection in a chorismate mutase deficient strain. An EcCM variant (Phe25', pBzF72, Thr76, Gly80' and Tyr83') forms a stable homodimer, has catalytic activity similar to the wild type enzyme, and unfolds with a T of 53 °C. The X-ray crystal structure reveals a pi-pi stacking and hydrogen bonding interactions that stabilize the new protein interface. The strategy described here should be useful for generating organisms that are dependent on the presence of a ncAA for growth.

PubMed: 28413876
DOI: 10.1021/jacs.7b02273

Experimental method

X-RAY DIFFRACTION (2 Å)