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Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure

DOI: 10.1038/s42003-019-0505-4 DOI Help
PMID: 31341959 PMID Help

Authors: Andani Mulelu (University of Cape Town) , Angela Kirykowicz (University of Cape Town) , Jeremy Woodward (University of Cape Town)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Communications Biology , VOL 2

State: Published (Approved)
Published: July 2019
Diamond Proposal Number(s): 20975

Open Access Open Access

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 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.

Journal Keywords: Biocatalysis; Electron microscopy; Hydrolases; Protein design; Secondary metabolism

Subject Areas: Biology and Bio-materials

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