Andani E Mulelu / Angela M Kirykowicz / Jeremy D Woodward /
PubMed Abstract
Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced ...Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.
EMDB-4418, PDB-6i5t: Cryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure. Method: EM (helical sym.) / Resolution: 3.9 Å
EMDB-4420, PDB-6i5u: Cryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure. Method: EM (helical sym.) / Resolution: 3.9 Å
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Keywords
arabidopsis thaliana (thale cress)