Chemistry Letters 2001
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suppressing the chitinolysis selectively, Chitinase A1 was mod-
ified to diminish the acidic character of E204, where the COOH
group was replaced with CONH2 group by site-specific muta-
tion from Glu to Gln (E204Q). Then, it was anticipated that the
mutant Chitinase A1 (ChiA1-E204Q) could catalyze the ring-
opening glycosylation of Chi2NAc-oxa exclusively.
Purified ChiA1-E204Q was prepared according to the pre-
vious procedure.5c ChiA1-E204Q (80 µg, 1.1 × 10–9 mol) was
added to a solution of Chi2NAc-oxa (8.5 mg, 2.1 × 10–5 mol) in
a phosphate buffer (pH 9.0, 40 mM, 125 µL) and incubated at
30 °C. The HPLC analysis indicated the formation of Chi4NAc
and Chi6NAc as the water-soluble product (Figure 1b).
Formation of GlcNAc, Chi3NAc and Chi5NAc was not detect-
ed. The concentrations of Chi4NAc and Chi6NAc increased
monotonously along with the reaction time and did not decrease
even after Chi2NAc-oxa disappeared. By addition of ChiA1-
Wt to the solution, however, the products were all hydrolyzed
into Chi2NAc and GlcNAc eventually. Similar results were
obtained by using six buffer solutions of Chi2NAc-oxa at differ-
ent pH values between 7.1–10.2. These results demonstrate
that ChiA1-E204Q still holds the catalytic activity for the ring-
opening glycosylation of Chi2NAc-oxa, while it is no more
active for chitinolysis.
2
3
4
5
a) T. Watanabe, K. Suzuki, W. Oyanagi, K. Ohnishi, and H.
Tanaka, J. Biol. Chem., 265, 15659 (1990). b) T. Watanabe,
W. Oyanagi, K. Suzuki, and H. Tanaka, J. Bacteriol., 172,
4017 (1990). c) T. Watanabe, K. Kobori, K. Miyashita, T.
Fujii, H. Sakai, M. Uchida, and H. Tanaka, J. Biol. Chem.,
268, 18567 (1993). d) T. Matsumoto, T. Nonaka, M.
Hashimoto, T. Watanabe, and Y. Mitsui, Proc. Japan. Acad.,
75, 269 (1999).
Little amount of water-insoluble products was produced by
using ChiA1-Wt. This result indicates that the polymeriza-
tion activity of ChiA1-Wt is much lower than that of the pre-
viously utilized chitinase (Bacillus sp.), which afforded poly-
saccharide chitin very efficiently as the major product.
a) A. C. Terwisscha van Scheltinga, S. Armand, K. H. Kalk,
A. Isogai, B. Henrissat, and B. W. Dijikstra, Biochemistry,
34, 15619 (1995). b) I. Tews, A. C. Terwisscha van
Scheltinga, A. Perrakis, K. S. Wilson, and B. W. Dijikstra, J.
Am. Chem. Soc., 119, 7954 (1997).
In conclusion, it has been demonstrated that the ring-open-
ing glycosylation of Chi2NAc-oxa is catalyzed by Chitinase A1
in the same active center with chitinolysis. The mechanistic
consideration on the catalytic function of E204 allowed to pre-
pare a nonchitinolytic mutant of Chitinase A1 for the exclusive
glycosylation of Chi2NAc-oxa. These results provide useful
information for in-depth understanding of the enzymatic cataly-
sis as well as clues for developing a new “glycosynthase”8 and
for the design of artificial substrates. Further studies are under
progress in our laboratories.
6
7
8
The authors thank Professor Shunsaku Kimura, Kyoto
University, for valuable suggestions and discussions.
References and Notes
1
a) S. Kobayashi, S. Shoda, and H. Uyama, in “Catalysis in
Precision Polymerization,” ed. by S. Kobayashi, John Wiley
& Sons, Chichester (1997), Chap. 8. b) S. Kobayashi and H.
Uyama, in “Materials Science and Technology – Synthesis of
Polymers,” ed. by A.-D. Schlüter, Wiley-VCH, Weinheim
(1998), Chap. 16. c) S. Kobayashi, J. Polym. Sci., Polym.
Chem. Ed., 37, 3041 (1999). d) S. Kobayashi, H. Uyama, and
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Schuelein, S. Cottaz, and H. Driguez, J. Am. Chem. Soc.,
122, 5429 (2000).