title: 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.
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhang
authors.forename: Bo
authors.initials: B
authors.lastname: Yin
authors.forename: Chao
authors.initials: C
authors.lastname: Wang
authors.forename: Shuiqin
authors.initials: S
authors.lastname: Jiang
authors.forename: Hualei
authors.initials: H
authors.lastname: Wang
authors.forename: Y Adam
authors.initials: YA
authors.lastname: Yuan
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000619:Aminohydrolases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D062105:Molecular Docking Simulation / Q000379:methods; D008969:Molecular Sequence Data; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D046939:Synechocystis / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D009570:Nitriles; D000619:Aminohydrolases; C021867:nitrilase; D003545:Cysteine