4F5H

Intercoversion of Substrate Specificity: E. coli Aspatate Aminotransferase to Tyrosine Aminotransferase: Chimera P3.

Summary for 4F5H

• Entry DOI: 10.2210/pdb4f5h/pdb
• Related: 4F5F 4F5G 4F5I 4F5J 4F5K 4F5L 4F5M
• Descriptor: Aspartate aminotransferase (2 entities in total)
• Functional Keywords: aminotransferase, transferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P00509
• Total number of polymer chains: 2
• Total formula weight: 89690.77

Authors

Addington, T.A.,Fisher, A.J.,Toney, M.D. (deposition date: 2012-05-13, release date: 2013-02-13, Last modification date: 2013-04-24)

Primary citation

Addington, T.A.,Mertz, R.W.,Siegel, J.B.,Thompson, J.M.,Fisher, A.J.,Filkov, V.,Fleischman, N.M.,Suen, A.A.,Zhang, C.,Toney, M.D.
Janus: prediction and ranking of mutations required for functional interconversion of enzymes.
J.Mol.Biol., 425:1378-1389, 2013

PubMed Abstract: Identification of residues responsible for functional specificity in enzymes is a challenging and important problem in protein chemistry. Active-site residues are generally easy to identify, but residues outside the active site are also important to catalysis and their identities and roles are more difficult to determine. We report a method based on analysis of multiple sequence alignments, embodied in our program Janus, for predicting mutations required to interconvert structurally related but functionally distinct enzymes. Conversion of aspartate aminotransferase into tyrosine aminotransferase is demonstrated and compared to previous efforts. Incorporation of 35 predicted mutations resulted in an enzyme with the desired substrate specificity but low catalytic activity. A single round of DNA back-shuffling with wild-type aspartate aminotransferase on this variant generated mutants with tyrosine aminotransferase activities better than those previously realized from rational design or directed evolution. Methods such as this, coupled with computational modeling, may prove invaluable in furthering our understanding of enzyme catalysis and engineering.

PubMed: 23396064
DOI: 10.1016/j.jmb.2013.01.034

Experimental method

X-RAY DIFFRACTION (1.6 Å)