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

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.

Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus

“…The homology model of NitTv1 was constructed previously [ 18 ]. Since then, a new template NIT4 in A. thaliana (pdb code 6i00) [ 21 ] emerged. In this study, this template was used for multiple sequence alignment ( Figure S3 ) along with the previously used template [ 18 ], i.e., the NLase from Synechocystis sp.…”

“…However, as this information on the active form of NitAb or NitTv1 has not been available yet, we used both the monomer and an oligomer (tetramer) for the docking studies ( Figures S7 and S8 in Supplementary Materials ). The recently reported structure of NIT4 [ 21 ] allowed a precise modeling of loop HL1 and hence improved prediction and characterization of ligand binding in the NLase oligomers.…”

“…The model of NitTv1 was previously constructed [ 18 ] and the model of NitAb was made analogously. The templates for homology modeling of NitAb were the recently released cryo-EM structure of NIT4 from A. thaliana (pdb code 6i00 [ 21 ]) and crystal structure of the NLase from Synechocystis sp. (pdb code 3wuy [ 22 ]).…”

“…The recent 6i00 structure allowed to refine modeling of loop HL1. The previously modeled NitTv1 structure was improved according to its closer homologue 6i00 [ 21 ]. Multiple sequence alignment was constructed with T-Coffee server [ 36 ] and used for NitAb model construction, and the refinement of the complete models and loops using MODELLER 9.16 [ 37 ] ( Appendix A ).…”

“…NLases usually form higher oligomers [ 3 ]. The oligomerization state of NitAb was proposed based on the best template—6i00 from Arabidopsis thaliana [ 21 ], and constructed by multiple structure alignment implemented in YASARA [ 40 , 51 ]. The proposed tetramer and dodecamer are shown in Figures S7 and S8A,B , respectively.…”