6432
J. Fang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6429–6432
Table 3
tary data associated with this article can be found, in the online
Conversion ratios of different sugar-1-phosphates with UTP in the reactions of full-
length GlmU and GlmU pyrophosphorylase domain
References and notes
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3. Kuhn, H. M.; Meier-Dieter, U.; Mayer, H. FEMS Microbiol. Rev. 1988, 4, 195.
4. Chang, R.; Moquist, P.; Finney, N. S. Carbohydr. Res. 2004, 339, 1531.
5. Herscovics, A.; Orlean, P. FASEB J. 1993, 7, 540.
6. Namboori, S. C.; Graham, D. E. J. Bacteriol. 2008, 190, 2987.
7. Levy, P.; Picard, J. Eur. J. Biochem. 1976, 61, 613.
GlmUa
88.3%
90.9%
74.2%
77.7%
34%
35.3%
GlmU-Tr229a
a
Enzyme concentration ꢁ 1.5 mg/100
l
L reaction system.
8. Feng, F.; Okuyama, K.; Niikura, K.; Ohta, T.; Sadamoto, R.; Monde, K.; Noguchi,
T.; Nishimura, S. Org. Biomol. Chem. 2004, 2, 1617.
9. Shao, J.; Zhang, J.; Nahálka, J.; Wang, P. G. Chem. Commun. 2002, 2586.
10. Milewski, S.; Gabriel, I.; Olchowy, J. Yeast 2006, 23, 1.
11. Mio, T.; Yabe, T.; Arisawa, M.; Yamada-Okabe, H. J. Biol. Chem. 1998, 273,
14392.
12. Mengin-Lecreulx, D.; van Heijenoort, J. J. Bacteriol. 1994, 176, 5788.
13. Bulter, T.; Elling, L. Glycoconjugate J. 1999, 16, 147.
14. Olsen, L. R.; Roderick, S. L. Biochemistry 2001, 40, 1913.
15. Mengin-Lecreulx, D.; van Heijenoort, J. J. Bacteriol. 1993, 175, 6150.
16. Parisi, G.; Echave, J. BMC Evol. Biol. 2004, 4, 41.
17. Brown, K.; Pompeo, F.; Dixon, S.; Mengin-Lecreulx, D.; Cambillau, C.; Bourne, Y.
EMBO J. 1999, 18, 4096.
18. Gehring, A. M.; Lees, W. J.; Mindiola, D. J.; Walsh, C. T.; Brown, E. D.
reaction in multiple mg scale for dUDP-GlcNAc and UDP-GlcNAcZ.
The reaction system contained 5 mM sugar-1-P and 5 mM NTP in a
final volume of 3 mL. Mono Q ion-exchange column (GE Health-
care) was used to isolate the products from the reaction mixture.
The column was eluted with a linear gradient of NaCl from 0 to
500 mM and the fractions containing sugar nucleotides were
pooled and concentrated. The products were further desalted by
P2 gel filtration column (Bio-rad). The isolated dUDP-GlcNAc
(5.1 mg, 57.6%) and UDP-GlcNAcZ (4.3 mg, 44.4%) were identified
by ESI-MS and NMR spectroscopy.20
N-Acetylglucosamine-1-phosphate uridyltransferase is re-
garded as an essential enzyme involved in prokaryote UDP-GlcNAc
biosynthetic pathway. Based on the previously reported crystal
structure, we cloned a truncated enzyme only with the pyrophos-
phorylase domain. The truncated GlmU showed substrate toler-
ance for both sugar-1-P and NTP, providing a feasible approach
for the synthesis of UDP-GlcNAc analogs. With this enzyme, two
UDP-GlcNAc derivatives, dUDP-GlcNAc and UDP-GlcNAcZ, were
in vitro synthesized. The former could be used to investigate the
NDP-sugar substrate specificity of glycosyltransferases, while the
latter could introduce an azide group into glycan structures, either
to facilitate glycosylation process detection or to be an intermedi-
ate for the synthesis of more complex glycan analogs with an ac-
tive group. Further broadening of the activity of this enzyme by
enzyme engineering based on its structure is ongoing in our lab.
Biochemistry 1996, 35, 579.
19. Cai, L.; Guan, W.; Kitaoka, M.; Shen, J.; Xia, C.; Chen, W.; Wang, P. G. Chem.
Commun. 2009, 2944.
20. Spectroscopic data of dUDP-GlcNAc: 1H NMR (600 MHz, D2O): d 7.79 (d, 1H,
J = 7.8 Hz, H-600), 6.16 (t, 1H, J = 6.6 Hz, H-10), 5.78 (d, 1H, J = 7.8 Hz, H-500), 5.34
(dd, 1H, J = 3.6, 7.2 Hz, H-1), 4.44 (m, 1H, H-30), 3.98–4.06 (m, 3H, H-40, H-
50a,b), 3.83 (dt, 1H, J = 3.6, 10.2 Hz, H-2), 3.76 (m, 1H, H-5), 3.71 (dd, 1H, J = 2.4,
12.6 Hz, H-6a), 3.65 (t, 1H, J = 10.2 Hz, H-3), 3.64 (dd, 1H, J = 4.2, 12.6 Hz, H-6b),
3.39 (t, 1H, J = 10.2 Hz, H-4), 2.20–2.25 (m, 2H, H-20a,b), 1.96 (s, 3H, Ac); 13C
NMR (150 MHz, D2O): d 174.6, 166.2, 151.5, 141.8 (C-600), 102.3 (C-500), 94.4 (C-
1), 85.4 (C-10), 85.3 (C-40), 72.9 (C-5), 70.8 (C-3), 70.7 (C-30), 69.3 (C-4), 65.3 (C-
50), 60.1 (C-6), 53.5 (d, C-2, J = 8.2 Hz), 38.7 (C-20), 21.9 (CH3CO). ESIMS
(negative ion): Calcd for C17H27N3O16P2: 591.09 [M]; Found: 590.2 [MꢀH]ꢀ.
Spectroscopic data of UDP-GlcNAcZ: 1H NMR (600 MHz, D2O): d 7.78 (d, 1H,
J = 8.4 Hz, H-600), 5.81 (d, 1H, J = 5.4 Hz, H-10), 5.79 (d, 1H, J = 8.4 Hz, H-500), 5.36
(dd, 1H, J = 3.0, 7.2 Hz, H-1), 4.17–4.22 (m, 2H, H-20, H-30), 4.11 (m, 1H, H-40),
4.07 (m, 1H, H-5a0), 4.01 (m, 1H, H-5b0), 3.97, 3.90 (2d, 2H, J = 16.2 Hz,
COCH2N3), 3.89 (dt, 1H, J = 3.0, 10.2 Hz, H-2), 3.76 (m, 1H, H-5), 3.70 (dd, 1H,
J = 2.4, 12.6 Hz, H-6a), 3.65 (t, 1H, J = 10.2 Hz, H-3), 3.64 (dd, 1H, J = 4.2,
12.6 Hz, H-6b), 3.39 (t, 1H, J = 10.2 Hz, H-4); 13C NMR (150 MHz, D2O): d 170.8,
166.2, 151.7, 141.5 (C-600), 102.5 (C-500), 94.2 (d, C-1, J = 6.0 Hz), 88.4 (C-10), 83.0
(d, C-40, J = 9.0 Hz), 73.6 (C-30), 72.9 (C-5), 70.7 (C-3), 69.5 (C-20), 69.3 (C-4),
64.9 (d, C-50, J = 6.0 Hz), 60.1 (C-6), 53.6 (d, C-2, J = 9.0 Hz), 51.4 (COCH2N3).
ESIMS (negative ion): Calcd for C17H26N6O17P2: 648.08 [M]; Found: 647.6
Supplementary data
Supplementary data contain the protein expression and purifi-
cation, CE profiles and ESI-MS data for substrates and products,
and NMR spectra for dUDP-GlcNAc and UDP-GlcNAcZ. Supplemen-
[MꢀH]ꢀ, 669.5 [Mꢀ2H+Na]ꢀ, 323.5 [Mꢀ2H]2ꢀ
.