Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

• Keywords
• Stenotrophomonas maltophilia, RNA, S1/P1 nuclease, X‐ray crystallography, c‐di‐GMP cleavage,
• MeSH
• Bacterial Proteins metabolism   chemistry   genetics   MeSH
• Cyclic GMP metabolism   analogs & derivatives   chemistry   MeSH
• Catalytic Domain * MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• RNA metabolism   chemistry   genetics   MeSH
• Stenotrophomonas maltophilia * enzymology   genetics   metabolism   MeSH
• Substrate Specificity MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Cyclic GMP MeSH
• RNA MeSH

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.

• MeSH
• Amino Acids metabolism   MeSH
• Aspergillus oryzae enzymology   metabolism   MeSH
• Single-Strand Specific DNA and RNA Endonucleases metabolism   MeSH
• Fungal Proteins metabolism   MeSH
• Catalytic Domain physiology   MeSH
• Catalysis MeSH
• Kinetics MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Substrate Specificity MeSH
• Binding Sites physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Endonuclease S1, Aspergillus MeSH Browser
• Single-Strand Specific DNA and RNA Endonucleases MeSH
• Fungal Proteins MeSH