Interestingly, the catalytic cores of SdaI and NotI are similar ( Figure 1A): six β strands of the central β sheets, including the conserved active site aspartate and glutamate residues, overlap between the NotI monomer and SdaI nucleolytic domain. This is in a sharp contrast to the NotI restriction enzyme, which combines insertions and metal binding domain into a compact single subunit which performs both catalysis and sequence recognition. Thus, SdaI employs two separate modules for catalysis and DNA sequence recognition. To interact with the 8 bp sequence, SdaI uses a wHTH DNA binding motif located in the N-terminal domain ( Figure 1B). The nuclease domain has a typical PD…(D/E)xK restriction endonuclease fold it has no loops or other structural elaborations that might be involved in the DNA sequence recognition.
SdaI is specific for the 8 bp sequence 5′-CCTGCA/GG-3′ and cuts it as designated by “/.” SdaI is composed of two domains: the N-terminal DNA-binding domain and the C-terminal nuclease domain ( Figure 1A). It would be interesting to see if the mutation of this His residue relaxes NotI specificity from 8 bp to 6 bp. No contacts are made to the neighboring cytosine residue. Interestingly, while contacts between NotI and the donor-acceptor atoms of the central six base pairs 5′-CGGCCG-3′ are fully saturated, those between the protein and the external GC base pair are limited to the single hydrogen bond between the His189 side chain and exocyclic carbonyl oxygen of the guanine.
In this respect, NotI is similar to a typical Type IIP restriction enzyme, which interacts with the palindromic nucleotide sequence. Other regions of the protein, including residues from a surface loop within the N-terminal iron-binding domain, also contribute to the DNA binding. Most of the residues involved in DNA sequence recognition are located within or close to the conserved nuclease core and are tightly coupled with the active site residues. The NotI enzyme approaches DNA from the major groove side and recognizes its 8 bp target sequence by making an extensive network of direct and water-mediated interactions to individual nucleotide bases.
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