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.
Highlights
Nucleases of the S1–P1 family [1, 2] can be found in fungi, plants, protozoan parasites and, interestingly, in some bacteria
We focus on the structure–function relationship in S1 nuclease, as a model system of the family, in complexes with varied ligands, in order to investigate substrate recognition, draw conclusions regarding its catalytic properties and provide structure–based background for design of inhibitors with potential applications in biotechnology and medicine
In six of the seven structures reported here at least one N–linked N– acetyl–D–glucosamine moiety (GlcNAc; the first saccharide unit left after deglycosylation using Endoglycosidase F1 (Endo F1)) participates in crystal contacts
Summary
Nucleases of the S1–P1 family [1, 2] can be found in fungi, plants, protozoan parasites and, interestingly, in some bacteria. They are zinc dependent nucleases/3’nucleotidases active on both RNA and DNA with acidic or close to neutral pH optima. A cluster of three zinc ions coordinates the substrate/product scissile phosphate and the reaction mechanism utilizes water activated by Zn2+ ions as a nucleophile [2] Their native role usually lies in nucleotides/nucleosides scavenging [3], specific apoptotic functions [4, 5] and in symbiont (pathogen)–host interactions [6]. Even if several studies already addressed ligand binding in this nuclease family exhaustive experimental evidence regarding ligand binding principles in this family is still missing
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