Endosialidases active site

Another feature common to all nonviral sialidases is the so-called Asp-box, a motif (S/T-X-D-[X]-G-X-T-W/F) that repeats one to five times along the sequences. Each Asp-box forms a clamp-like loop and they occur at topologically equivalent positions on the outside of the structure between the third and fourth p-strand of a propeller blade [12, 114, 115]. For example, in the sialidase Nani of Clostridium perfringens four Asp-boxes are located in the blades one to four (PDB ID 2BF6 [115]). However, in endosialidases only two Asp-boxes have been found in the first and fourth blade of the propeller [12], with the sequences S-G-D-D-G-Q/ K-T-W and S-X-D-X-G-X-X-W that are conserved in aU so far known endosialidases. Interestingly, in endoNF the p-barrel domain is inserted between the third and fourth p-sheet of the second blade, thereby replacing a potential Asp-box. Since the Asp-boxes are located on the back side of the propeller and remote from the active site, any functions other than structural folds have not been found as yet for these motifs. 

As discussed in the previous section, exosialidases and endosialidases have different active site topologies. Although three residues have been identified to be highly... 

Tyr-325 (b). After release of the reducing end moiety (ROH, c) the initial state of the active site is regenerated by proton transfer via a water chain ([H20] , d) and re-binding of polysiahc acid. In total, the stmctural data allow the conclusion that the endosialidase mechanism is an SNl-type reaction, i.e., the sialic acid stereochemistry is directly inverted into the p-anomer [106]. By contrast, exosialidases form a covalently bound intermediate which is released by a water molecule thus the a2,8-linked sialic acid residue is released as a-anomer tmderlying the mutarotation towards the energetically favored p-conformation. However, no crystal structure of a non-cleavable substrate is available as yet, which would allow an unambiguous elucidation of the endosialidase mechanism [106]. 

The aligned sequences of the endosialidases of phages KIA and KIF (endoNF) have 83% identity [69], with the major domains having even higher identity ((3-propeller sequences 93% and the p-barrels 100%). A homology-based structural model of the endosiahdases predicts the spatial arrangement of the mutant amino acids close to each other at the active site of the enzyme [69] (Fig. 2b). The mutations affect the orientation of the residues and the shape of the active site cleft. 

Jakobsson E, Jokilammi A, Aalto J, Ollikka P, Lehtonen IV, Hirvonen H, Finne J (2007) Identification of amino acid residues at the active site of endosialidase that dissociate the poly sialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages. Biochem J 405 465-472... 

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