6i00

Publication Abstract from PubMed

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 A) 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 alpha2 and alpha3 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.

Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure.,Mulelu AE, Kirykowicz AM, Woodward JD Commun Biol. 2019 Jul 17;2:260. doi: 10.1038/s42003-019-0505-4. eCollection 2019. PMID:31341959^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.