Keratanase II-catalyzed synthesis of keratan sulfate oligomers by using sugar oxazolines as transition-state analogue substrate monomers: a novel insight into the enzymatic catalysis mechanism.
Keratan sulfate (KS) oligomers with well-defined structures were synthesized by keratanase II (KSase II)-catalyzed transglycosylation. N-Acetyllactosamine [Galbeta(1-->4)GlcNAc; LacNAc] oxazoline derivatives with sulfate groups at the C-6 (1 a) and both the C-6 and the C-6' (1 b) were prepared as transition-state analogue substrate monomers for KSase II. Monomer 1 a was effectively oligomerized by the enzyme under weak alkaline conditions, to give alternating 6-sulfated KS oligomers (2 a) in good yields, and with total control of regioselectivity and stereochemistry. KSase II also recognized 1 b, which provided fully 6-sulfated KS oligomers (2 b) in good yields under similar conditions. Nonsulfated LacNAc oxazoline was difficult to oligomerize enzymatically. These results imply that the catalysis mechanism of KSase II involves a sugar oxazolinium ion that requires the 6-sulfate group in the GlcNAc residue not only in hydrolysis of KS chains, but also in oligomerization of oxazoline monomers. This is the first report of KSase II-catalyzed transglycosylation to form beta(1-->3)-glycosidic bond through a substrate-assisted mechanism.
Ohmae
Masashi
M
Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan. ohmae@peptide.polym.kyoto-u.ac.jp
Sakaguchi
Kazuya
K
Kaneto
Taihei
T
Fujikawa
Shun-ichi
S
Kobayashi
Shiro
S
D000118:Acetylglucosaminidase / Q000737:chemistry*; D020816:Amino Acid Motifs; D000596:Amino Acids / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002384:Catalysis; D002845:Chromatography / Q000379:methods; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007632:Keratan Sulfate / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013997:Time Factors
D000596:Amino Acids; D010080:Oxazoles; D007632:Keratan Sulfate; C086950:keratanase II; D000118:Acetylglucosaminidase