LAUSR

The reaction specificity of the transglycosylation catalyzed by keratanase II from Bacillus circulans KsT202 (KSase II) was studied by using the oxazoline derivatives having keratan sulfate repeating units. The addition of 10% organic cosolvent reduced the activity for the enzymatic transglycosylation. The oxazoline derivative of 6-O-sulfonato-N-acetyllactosamine (su-LacNAc) was processively oligomerized to the corresponding hexamer or longer by the enzyme. This result strongly implies that the enzyme has the large positively numbered subsites. In contrast, the transglycosylation of the su-LacNAc oxazoline donor with the 6-O-sulfonato-Lewis X (su-LeX) acceptor solely gave the su-LacNAc-su-LeX pentasaccharide. In addition, both the oxazoline derivatives of su-LeX and 6,6'-di-O-sulfonato-LacNAc have been exclusively oligomerized to the corresponding dimers respectively. These results strongly suggest that the steric hindrance exists around the (+3)(+4) subsites in KSase II. Furthermore, KSase II-catalyzed reaction of the excess su-LeX oxazoline with the su-LacNAc gave the su-LeX-su-LacNAc pentasaccharide as the sole transglycosylation product, also implying the steric hindrance at the catalytic center hampering processive shift of this pentasaccharide. Thus, KSase II has the sterically crowded structures at the catalytic center and around the (+3)(+4) subsites, which are all expected to be tunnel-like.