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.

• Klíčová slova
• Stenotrophomonas maltophilia, RNA, S1/P1 nuclease, X‐ray crystallography, c‐di‐GMP cleavage,
• MeSH
• bakteriální proteiny metabolismus   chemie   genetika   MeSH
• guanosinmonofosfát cyklický metabolismus   analogy a deriváty   chemie   MeSH
• katalytická doména * MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• RNA metabolismus   chemie   genetika   MeSH
• Stenotrophomonas maltophilia * enzymologie   genetika   metabolismus   MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• guanosinmonofosfát cyklický 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
• aminokyseliny metabolismus   MeSH
• Aspergillus oryzae enzymologie   metabolismus   MeSH
• endonukleasy specifické pro jednořetězcové nukleové kyseliny metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• katalytická doména fyziologie   MeSH
• katalýza MeSH
• kinetika MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• substrátová specifita MeSH
• vazebná místa fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• Endonuclease S1, Aspergillus MeSH Prohlížeč
• endonukleasy specifické pro jednořetězcové nukleové kyseliny MeSH
• fungální proteiny MeSH

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

• Klíčová slova
• TBN1, superhelix, tomato multifunctional nuclease, type I nuclease,
• MeSH
• endodeoxyribonukleasy chemie   genetika   metabolismus   MeSH
• fosfáty metabolismus   MeSH
• krystalizace MeSH
• molekulární sekvence - údaje MeSH
• multienzymové komplexy chemie   genetika   metabolismus   MeSH
• rostlinné proteiny chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Solanum lycopersicum enzymologie   genetika   MeSH
• vazebná místa fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endodeoxyribonukleasy MeSH
• fosfáty MeSH
• multienzymové komplexy MeSH
• rostlinné proteiny MeSH
• superhelical DNA endonuclease MeSH Prohlížeč