Reaction specificity of keratanase II in the transglycosylation using the sugar oxazolines having keratan sulfate repeating units. (1st February 2018)

• Record Type: Journal Article
• Authors: Yamazaki, Yuji; Kimura, Shunsaku; Ohmae, Masashi
• Is Part Of: Carbohydrate research. Volume 456(2018)
• Page Start: 61
• Page End: 68
• Publication Date: 2018-02-01
• Subjects: Enzymatic glycosylation -- Sugar oxazolines -- Keratanase II -- Subsite mapping -- Substrate specificity -- Keratan sulfate; Carbohydrates -- Periodicals; Chemistry, Organic -- Periodicals; Biochemistry -- Periodicals; Carbohydrates -- Periodicals; Chimie organique -- Périodiques; Glucides -- Périodiques; Biochemistry; Carbohydrates; Chemistry, Organic; Periodicals; Electronic journals; 507.78
• DOI: 10.1016/j.carres.2017.12.003
• Languages: English
• ISSNs: 0008-6215

Abstract

Abstract: 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-Le X ) acceptor solely gave the su-LacNAc-su-Le X pentasaccharide. In addition, both the oxazoline derivatives of su-Le X 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-Le X oxazoline with the su-LacNAc gave the su-Le X -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. Graphical abstract: Highlights: Keratanase II (KSase II) is highly sensitive to the addition of organic solvents. KSase II can catalyze processive transglycosylation to keratan sulfate oligomers. KSase II has at least the six subsites of (−2)(−1)(+1)(+2)(+3)(+4). Sterically hindered points exist near the catalytic center and the (+3)(+4) subsites. The structures of the two sterically hindered points are supposed to be tunnel-like.