1148; (c) I. M. Saxena and R. M. Brown, Jr., Curr. Opp. Plant Biol.,
Dr Herman van Halbeek, Mr Steven Adams and Mr Dennis
Koehler (UCSD) for technical assistance, and Professors Scott
Singleton (Rice University) and Jack Kyte (UCSD) for helpful
discussions.
2000, 3, 523–531; (d ) I. M. Saxena, R. M. Brown, Jr., M. Fevre,
R. A. Geremia and B. Henrissat, J. Bacteriol., 1995, 177, 1419–1424.
7 It has been shown that the growing chitin chain is extended from the
non-reducing terminus: J. Sugiyama, C. Boisset, M. Hashimoto and
T. Watanabe, J. Mol. Biol., 1999, 286, 247–255.
8 More important than the energetic cost of the distortion is the fact
that the transition states leading to the “normal” and “distorted”
intermediates would have very different geometries, both of which
would have to be stabilized by a common set of active-site functional
groups.
References and Notes
1 Representative reviews of fungal chitin synthase (EC 2.4.1.16).
(a) C. A. Munro and N. A. R. Gow, Med. Mycol., 2001, 39, 41–53;
(b) M. H. Valdivieso, A. Duran and C. Roncero, EXS, 1999, 87,
55–69; (c) R. A. Merz, M. Horsch, L. E. Nyhlen and D. M. Rast,
EXS, 1999, 87, 9–37; (d ) C. E. Bulawa, Ann. Rev. Microbiol., 1993,
47, 505–534; (e) E. Cabib, Advances Enzymol., 1987, 59, 59–101.
2 The significance of glycosyltransferases derives from the fact that
mono- or polysaccharides are involved in virtually all biological
processes. For an excellent overview of the field, see (a) Essentials
of Glycobiology, eds. A. Varki, R., Cummings, J. Esko, H. Freeze,
G. Hart and J. Marth, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1999.; For recent reviews; (b) R. A. Dwek
and T. D. Butters, Chem. Rev., 2002, 102, 283–284; (c) C. R. Bertozzi
and L. L. Kiessling, Science, 2001, 291, 2357–2364; (d ) A. Dove,
Nature Biotechnol., 2001, 19, 913–917; (e) A. Kobata, Glycoconju-
gate J., 2001, 17, 443–464; ( f ) K. M. Koeller and C.-H. Wong,
Nature Biotechnol., 2000, 18, 835–841.
9 H. Kazahura, S. Nisimura, K. Shimahara and Y. Takiguchi,
Carbohydr. Res., 1989, 194, 223–231.
10 All new compounds were fully characterized by 1H- and 13C-NMR,
IR and high-resolution mass spectrometry. Experimental details are
available upon request.
11 J. K. Coward and J. Lee, J. Org. Chem., 1992, 57, 4126–4135.
12 H. G. Khorana and A. R. Todd, J. Chem. Soc., 1953, 2257–
2260.
13 (a) C.-H. Wong and V. Wittman, J. Org. Chem., 1997, 57, 2144–2147;
(b) J. G. Moffatt, Methods Enzymol., 1966, 8, 136–142.
14 Y. Eshdat and N. Sharon, Methods Enzymol., 1977, 46, 403–414.
15 P. Orlean, J. Biol. Chem., 1987, 262, 5732–5739 . While the original
procedure employed 14C-UDP-GlcNAc, we found the less-expensive
3H-UDP-GlcNAc to be an acceptable substitute.
3 While natural product inhibitors are known, their effectiveness has
been limited by low affinity and selectivity. The closely-related poly-
oxins and nikkomycins are among the most potent inhibitors, with
Ki values in the low mM range. None of these have sufficient in vivo,
activity for therapeutic use, although polyoxin D is used as an agri-
cultural fungicide. See: C. A. Kauffman and P. L. Carver, Drugs,
1997, 53, 539–49.
4 For recent reviews of mechanism-based inhibitors of glycosidases
and non-processive glycosyltransferase, see (a) P. Compain and
O. R. Martin, Bioorg. Med. Chem., 2001, 99, 3077–3092; (b) P. Sears
and C.-H. Wong, Angew. Chem., Int. Ed., 1999, 38, 2300–2324.
5 Several crystal structures of non-processive transferases have been
determined. For discussion of common features, see (a) G. J. Davies,
Nature Struct. Biol., 2001, 8, 98–100; (b) K. Persson, H. D. Ly,
M. Dieckelmann, W. W. Wakarchuk, S. G. Withers and N. C. J.
Strynadka, Nature Struct. Biol., 2001, 8, 166–175; (c) S. J. Charnok,
B. Henrissat and G. J. Davies, Plant Physiol., 2001, 125, 527–531.
6 Representative mechanistic discussion (a) refs 5a,c; (b) I. M. Saxena,
R. M. Brown, Jr. and T. Dandekar, Phytochemistry, 2001, 57, 1135–
16 Chitin synthase does not require an initial acceptor other than
UDP-GlcNAc to initiate polymerization. (See refs 1,6.). The fate of
the UDP fragment from the initiating sugar is unclear, and thus a
unique requirement for UDP-GlcNAc as initiator could not be
excluded a priori.
17 Ki values were obtained from the relationship Ki = IC50/(1ϩ[sub-
strate]/Km) (ref. 18). Assays were performed at [UDP-GlcNAc] =
1.0 mM. KM = 0.5 mM, so this reduces to Ki = IC50/3. IC50 was
determined by non-linear least-squares fitting of a plot of inhibition
(%) vs. log [UDP-Chi] (measured at [UDP-GlcNAc] = 1 mM. using
the Prism3 software package (Graphpad Inc., San Diego, CA); log
IC50 for UDP-Chi was determined to be 1.0 0.1 (95% confidence
limits), providing IC50 = 10.0 ϩ2.6/Ϫ2.1 (7.9 Ϫ 12.6).
18 Y. Cheng and W. H. Prusoff, Biochem. Pharmacol., 1973, 22, 3099–
3108.
19 I. H. Segel, Enzyme Kinetics, John Wiley & Sons, New York, NY,
1975 .
20 The method of Scheme 1 has been used to prepare UDP-cellobiose.
R. Chang, N. S. Finney, unpublished results.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 – 4 1
41