Janus: Prediction and ranking of mutations required for functional interconversion of enzymes

Trevor A. Addington, Robert W. Mertz, Justin Siegel, James M. Thompson, Andrew J Fisher, Vladimir Filkov, Nicholas M. Fleischman, Alisa A. Suen, Chensong Zhang, Michael D. Toney

Research output: Contribution to journalArticle

14 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1378-1389
Number of pages12
JournalJournal of Molecular Biology
Volume425
Issue number8
DOIs
StatePublished - Apr 26 2013

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Keywords

  • bioinformatics
  • computational modeling
  • directed evolution
  • enzymology
  • protein engineering

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Addington, T. A., Mertz, R. W., Siegel, J., Thompson, J. M., Fisher, A. J., Filkov, V., Fleischman, N. M., Suen, A. A., Zhang, C., & Toney, M. D. (2013). Janus: Prediction and ranking of mutations required for functional interconversion of enzymes. Journal of Molecular Biology, 425(8), 1378-1389. https://doi.org/10.1016/j.jmb.2013.01.034