284
T. Yang and others
identify how subtle changes in amino acids allow it to be able
to accept the C4-epimer (i.e. gluco- compared with galacto-
configurations). Comparison of the homology model between
GlcNAc1pUT-1 and GlcNAc1pUT-2 indicated that both enzymes
share a similar fold, except for some variations in some loop
region (Figure 5B). The loop in approximation to the sugar moiety
8
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Silverstone, A. L., Tseng, T. S., Swain, S. M., Dill, A., Jeong, S. Y., Olszewski, N. E. and
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317
between amino acids Pro and Gly on GlcNAc1pUT-2 appears
different to GlcNAc1pUT-1. Whether this is the actual cause
of the GlcNAc1pUT-2 to take UDP-Glc as a substrate remains
unknown. Thecrystalstructure isrequired to solve thepromiscuity
of GlcNAc1pUT-2. The promiscuity of GlcNAc1pUT-2 to both
Glc-1-P and GlcNAc-1-P as substrates is not a unique feature of
Arabidopsis, as the yeast PPase [15] utilizes both substrates as
well.
The biological significance of GlcNAc1p-UT is critical, not
only as post-translational modification of regulatory proteins,
but also as a component of glycolipid and glycoprotein. So far
T-DNA insertion in At1g31070 loci has no visible phenotype
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14 Alonso, A. P., Piasecki, R. J., Wang, Y., Laclair, R. W. and Shachar-Hill, Y. (2010)
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chromatography tandem mass spectrometry. Plant Physiol. 153, 915–924
(
T. Yang, unpublished work). Considering the key role of UDP-
GlcNAc in development and regulation, and the fact that two genes
encoding this activity appears to be expressed in most Arabidopsis
tissues, suggest that both genes need to be knockout for functional
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yeast. Yeast 23, 1–14
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1
1
1
1
6
7
8
9
AUTHOR CONTRIBUTION
Ting Yang was involved in all aspects of the study, including experimental design,
performing the research, data analysis and manuscript preparation. Merritt Echols and
Andy Martin were involved in performing the research. Maor Bar-Peled directed the study
and was involved in all aspects of experimental design, data analysis and manuscript
revision.
20, 6191–6202
2
2
0
1
Yang, T., Bar-Peled, L., Gebhart, L., Lee, S. G. and Bar-Peled, M. (2009) Identification of
galacturonic acid-1-phosphate kinase, a new member of the GHMP kinase superfamily in
plants, and comparison with galactose-1-phosphate kinase. J. Biol. Chem. 284,
ACKNOWLEDGEMENTS
We thank Dr John Glushka for his guidance and assistance with the NMR experiments.
2
1526–21535
Kelley, L. A. and Sternberg, M. J. (2009) Protein structure prediction on the Web: a case
study using the Phyre server. Nat. Protoc. 4, 363–371
FUNDING
This work was supported by the National Science Foundation [grant number IOB-0453664
22 Arnold, K., Bordoli, L., Kopp, J. and Schwede, T. (2006) The SWISS-MODEL workspace:
(
to M.B.-P.)] and the BioEnergy Science Center, which is supported by the Office of
a web-based environment for protein structure homology modelling. Bioinformatics 22,
Biological and Environmental Research in the DOE Office of Science. This research also
benefited from activities at the Southeast Collaboratory for High-Field Biomolecular NMR,
a research resource at the University of Georgia, funded by the National Institute of General
Medical Sciences [grant number GM66340] and the Georgia Research Alliance.
1
95–201
2
2
3
4
McCoy, J. G., Bitto, E., Bingman, C. A., Wesenberg, G. E., Bannen, R. M., Kondrashov,
D. A. and Phillips, Jr, G. N. (2007) Structure and dynamics of UDP-glucose
pyrophosphorylase from Arabidopsis thaliana with bound UDP-glucose and UTP. J. Mol.
Biol. 366, 830–841
Meng, M., Wilczynska, M. and Kleczkowski, L. A. (2008) Molecular and kinetic
characterization of two UDP-glucose pyrophosphorylases, products of distinct genes,
from Arabidopsis. Biochim. Biophys. Acta 1784, 967–972
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Received 1 March 2010/15 June 2010; accepted 17 June 2010
Published as BJ Immediate Publication 17 June 2010, doi:10.1042/BJ20100315
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society