Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803

Abstract

Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys–Glu–Lys catalytic triad to hydrolyze non-peptide carbon–nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 Å resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates.

Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of ( R )- o -Chloromandelic Acid

“…The first step of nucleophilic attack that is mediated by C164 is crucial for determining the hydrolysis efficiency. A sufficiently small through-space distance (d) value between the nucleophilic sulfur of C164 and the cyano carbon atom of the substrate could correspond to a near-attack conformer, as discussed previously for nitrilase and other enzymecatalyzed reactions (14,23), and this distance should be shorter in the case of the preferred enantiomer.…”

“…Five nitrilase crystal structures with the highest homology to BCJ2315 were chosen as the templates: Nit6803 (PDB accession number 3WUY, 57% similarity and 36% identity) from Synechocystis sp. strain PCC6803 (14), PaNit (PDB accession number 3IVZ, 42% similarity and 25% identity) from Pyrococcus abyssi (15), hypothetical protein Ph0642 (PDB accession number 1J31, 41% similarity and 25% identity) from Pyrococcus horikoshii (16), mouse nitrilase 2 (PDB accession number 2W1V, 41% similarity and 23% identity) (17), and Nit3 (PDB accession number 1F89, 38% similarity and 25% identity) from Saccharomyces cerevisiae (18). The structure ranking was based on the probability density function (PDF) total energy and evaluated by Verify Protein (Profiles-3D) and Ramachandran plots in DS 4.1.…”

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

“…The three‐dimensional (3D) structures of the parent nitrilases and variants were constructed via the automated protein modeling server SWISS‐MODEL (http://swissmodel.expasy.org/; Arnold, Bordoli, Kopp, & Schwede, ) on the basis of homology modeling with reference to the crystal structure of Syechocystis sp. PCC6803 nitrilase (Nit6803, PDB ID: 3WUY; Zhang et al, ), which shared the highest sequence identity to the parent nitrilases.…”

“…The three-dimensional (3D) structures of the parent nitrilases and variants were constructed via the automated protein modeling server SWISS-MODEL (http://swissmodel.expasy.org/;Arnold, Bordoli, Kopp, & Schwede, 2006) on the basis of homology modeling with reference to the crystal structure of Syechocystis sp. PCC6803 nitrilase (Nit6803, PDB ID: 3WUY;Zhang et al, 2014), which shared the highest sequence identity to the parent nitrilases.The MD simulations for the parent nitrilases and variants were performed using GROMACS version 4.0 and OPLS-AA force field. Each nitrilase was placed in the center of a cubic box consisting of SPC216 water molecules, with periodic boundary conditions.…”

Interplay of nucleophilic catalysis with proton transfer in the nitrile reductase QueF from Escherichia coli

“…[1][2][3] They also have significant applications in synthetic chemistry wherein nitriles often represent important intermediates. 4,5 The nitrile group is converted by hydrolytic enzymes to an amide or to a carboxylic acid [6][7][8][9] and by reductive enzymes to an amine. [10][11][12][13] Despite the different reactivities, nitrile-converting enzymes share covalent catalysis from an active-site nucleophile as a common feature of their mechanisms.…”