title: Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non-Specificity, and Inhibition.
abstract: The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Kovaľ
authors.affiliation: Department of Agile Protein Screening, Novozymes A/S, Bagsvaerd, Denmark.
authors.forename: Lars H
authors.initials: LH
authors.lastname: Østergaard
authors.affiliation: Department of Fungal Strain Technology, Novozymes A/S, Bagsvaerd, Denmark.
authors.forename: Jan
authors.initials: J
authors.lastname: Lehmbeck
authors.affiliation: Department of Protein Biochemistry and Stability, Novozymes A/S, Bagsvaerd, Denmark.
authors.forename: Allan
authors.initials: A
authors.lastname: Nørgaard
authors.affiliation: Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic.
authors.forename: Petra
authors.initials: P
authors.lastname: Lipovová
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Jarmila
authors.initials: J
authors.lastname: Dušková
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Tereza
authors.initials: T
authors.lastname: Skálová
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Mária
authors.initials: M
authors.lastname: Trundová
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Kolenko
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Karla
authors.initials: K
authors.lastname: Fejfarová
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Stránský
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Leona
authors.initials: L
authors.lastname: Švecová
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Jindřich
authors.initials: J
authors.lastname: Hašek
authors.affiliation: Laboratory of Structure and Function of Biomolecules, Institute of Biotechnology CAS, v. v. i., Biocev, Vestec, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Dohnálek
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology; D005656:Fungal Proteins / Q000378:metabolism*; D007700:Kinetics; D016415:Sequence Alignment; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; C526098:Endonuclease S1, Aspergillus; D015719:Single-Strand Specific DNA and RNA Endonucleases