6I00

Cryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure.

Summary for 6I00

• Entry DOI: 10.2210/pdb6i00/pdb
• EMDB information: 0320
• Descriptor: Bifunctional nitrilase/nitrile hydratase NIT4 (1 entity in total)
• Functional Keywords: nitrilase, cyanide detoxification, beta-cyano-l-alanine hydrolase, helical filament, hydrolase
• Biological source: Arabidopsis thaliana (Mouse-ear cress)
• Total number of polymer chains: 12
• Total formula weight: 477193.36

Authors

Mulelu, A.E.,Woodward, J.D. (deposition date: 2018-10-24, release date: 2019-07-24, Last modification date: 2024-05-15)

Primary citation

Mulelu, A.E.,Kirykowicz, A.M.,Woodward, J.D.
Cryo-EM and directed evolution reveal howArabidopsisnitrilase specificity is influenced by its quaternary structure.
Commun Biol, 2:260-260, 2019

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 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 . 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.

PubMed: 31341959
DOI: 10.1038/s42003-019-0505-4

Experimental method

ELECTRON MICROSCOPY (3.4 Å)