Janus: prediction and ranking of mutations required for functional interconversion of enzymes.

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.

[1]  Christina Gloeckner,et al.  Modern Applied Statistics With S , 2003 .

[2]  S. Primrose,et al.  The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes. , 1986, The Biochemical journal.

[3]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[4]  H Hayashi,et al.  Crystal structures of Paracoccus denitrificans aromatic amino acid aminotransferase: a substrate recognition site constructed by rearrangement of hydrogen bond network. , 1998, Journal of molecular biology.

[5]  Oliver F. Lange,et al.  NMR Structure Determination for Larger Proteins Using Backbone-Only Data , 2010, Science.

[6]  David Baker,et al.  De Novo Enzyme Design Using Rosetta3 , 2011, PloS one.

[7]  Jack F Kirsch,et al.  How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase. , 2003, Journal of molecular biology.

[8]  Anna R Panchenko,et al.  Coevolution in defining the functional specificity , 2009, Proteins.

[9]  H. Hayashi,et al.  Escherichia coli aromatic amino acid aminotransferase: characterization and comparison with aspartate aminotransferase. , 1993, Biochemistry.

[10]  Jens Meiler,et al.  ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. , 2011, Methods in enzymology.

[11]  J. Kirsch,et al.  Decreasing the basicity of the active site base, Lys-258, of Escherichia coli aspartate aminotransferase by replacement with gamma-thialysine. , 1995, Biochemistry.

[12]  H. Hayashi,et al.  Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity. , 1990, Biochemistry.

[13]  R. Ranganathan,et al.  Evolutionarily conserved pathways of energetic connectivity in protein families. , 1999, Science.

[14]  David Baker,et al.  An exciting but challenging road ahead for computational enzyme design , 2010, Protein science : a publication of the Protein Society.

[15]  N. Goodey,et al.  Catalytic contributions from remote regions of enzyme structure. , 2011, Chemical reviews.

[16]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[17]  F. Cohen,et al.  An evolutionary trace method defines binding surfaces common to protein families. , 1996, Journal of molecular biology.

[18]  J. Kirsch,et al.  Quantitative chimeric analysis of six specificity determinants that differentiate Escherichia coli aspartate from tyrosine aminotransferase , 2002, Protein science : a publication of the Protein Society.

[19]  SödingJohannes.  Protein homology detection by HMM--HMM comparison , 2005 .

[20]  J. Jansonius,et al.  Crystal structures of Escherichia coli aspartate aminotransferase in two conformations. Comparison of an unliganded open and two liganded closed forms. , 1994, Journal of molecular biology.

[21]  Mona Singh,et al.  Characterization and prediction of residues determining protein functional specificity , 2008, Bioinform..

[22]  J. Kirsch,et al.  Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase , 2004, Protein science : a publication of the Protein Society.

[23]  Vladimir A. Ivanisenko,et al.  WebProAnalyst: an interactive tool for analysis of quantitative structure–activity relationships in protein families , 2005, Nucleic Acids Res..

[24]  J. Kirsch,et al.  A general method for the quantitative analysis of functional chimeras: Applications from site‐directed mutagenesis and macromolecular association , 2001, Protein science : a publication of the Protein Society.

[25]  Tsuneo Yamane,et al.  Chimeric Gene Library Construction by a Simple and Highly Versatile Method Using Recombination‐Dependent Exponential Amplification , 2003, Biotechnology progress.

[26]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[27]  David Baker,et al.  Incorporation of evolutionary information into Rosetta comparative modeling , 2011, Proteins.

[28]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[29]  Mikhail S. Gelfand,et al.  SDPpred: a tool for prediction of amino acid residues that determine differences in functional specificity of homologous proteins , 2004, Nucleic Acids Res..

[30]  Nikolay A. Kolchanov,et al.  CRASP: a program for analysis of coordinated substitutions in multiple alignments of protein sequences , 2004, Nucleic Acids Res..

[31]  J. Kirsch,et al.  Redesign of the substrate specificity of escherichia coli aspartate aminotransferase to that of escherichia coli tyrosine aminotransferase by homology modeling and site‐directed mutagenesis , 1995, Protein science : a publication of the Protein Society.

[32]  M. Kula,et al.  d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei , 2004, Applied Microbiology and Biotechnology.

[33]  P. Christen,et al.  Changing the reaction specificity of a pyridoxal-5'-phosphate-dependent enzyme. , 1995, European journal of biochemistry.

[34]  V. Malashkevich,et al.  Alternating arginine-modulated substrate specificity in an engineered tyrosine aminotransferase , 1995, Nature Structural Biology.

[35]  John M Joern,et al.  DNA shuffling. , 2003, Methods in molecular biology.

[36]  Jack F Kirsch,et al.  Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase. , 2004, Biochemistry.

[37]  John C Whitman,et al.  Improving catalytic function by ProSAR-driven enzyme evolution , 2007, Nature Biotechnology.