Substrate specificity of plant nitrilase complexes is affected by their helical twist

Abstract

Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar nitrilases from the plant Capsella rubella, that modifying nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.

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

“…Branch 1 nitrilases (EC 3.5.5.1) (NITs) are industrially important 1 oligomeric enzymes (forming left-handed twists, spirals, turns or helices) that share a common architecture 2–8 and catalyze the hydrolysis of nitriles to carboxylic acids and ammonia and/or amides 9 . In all plants, the nitrilase 4 (EC 4.2.1.65) (NIT4) group represents the ancestral form, which is highly specific for β-cyano- l -alanine 10 .…”

“…It is thought that the NIT1 group enzymes evolved to catabolise the diverse glucosinolate-derived nitriles found in the Brassicaceae 11–13 . Interestingly, plant NITs with high specificity against a single substrate or narrow group of substrates have evolved from the ancestral generalist NIT1 group enzymes 8,13,14 .…”

“…We recently discovered that substrate specificity in plant NITs is related to the structure of the NIT helical assembly 8 . The helical twist (Δ ϕ ) of the NIT filament is correlated with the size of the preferred substrate: NIT4 enzymes have a relatively large helical twist, small substrate size, and high specificity, while NIT1 enzymes have a smaller twist and broader specificity.…”

Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor

“…C surface is asymmetric interaction between two dimers, which plays an essential role in the formation of the spiral quaternary structure of nitrilases. It was reported that substitution of residues in the A and C surface could greatly alter the substrate specificity, thermostability, and activity of nitrilase (Sewell et al, ; Woodward, Trompetter, Sewell, & Piotrowski, ; Xu et al, ). The catalytic triads of Aa NIT and Br NIT were located at positions 65 and 66 (Glu), 152 and 153 (Lys), and 186 and 187 (Cys), respectively.…”

“…The flexible loops on the C surface of the wild-type BrNIT were replaced by a helix (residues 242-256) of BaNIT (as illustrated in Figure 5c). As the A and C surface play critical roles in the formation of the spiral quaternary structure of nitrilases (Thuku, Brady, Benedik, & Sewell, 2009), these changes may affect the conformation of higher-order spiral structure. What is more, a helix (residues 188-191) located near the catalytic residue Cys187 of the wild-type BrNIT disappeared in the chimera BaNIT (Figure 5b).…”