Yeast chorismate mutase as a model system for studying allostery through NMR
Scott D. Gorman, Debashish Sahu and David D. Boehr
Department of Chemistry, The Pennsylvania State University, University Park, PA
The AroQ family of chorimase mutases consists of all-alpha helical
enzymes that convert chorismate to prephenate.1 This reaction is an
important step in the production of phenylalanine and tyrosine in the
shikimic acid pathway, which is absent in animals but present in
bacteria, fungi, and plants.2 There are also examples of secreted
AroQ chorismate mutases that are thought to be involved in hostpathogen interactions in bacteria and parasitic animals that are not
part of the shikimic acid pathway.3,4 Thus a better understanding of
chorismate mutases would potentially be beneficial to agriculture,
medicine, and industry.
Figure 1. ScCM homodimer with the transition state
analogue bound in turquoise. The regulatory domain
of the left monomer is show in magenta, and the
catalytic domain in violet. PDB: 3CSM.
Figure 2. 1H-15N HSQC of ScCM with tyrosine
bound.
Saccharomyces cerevisiae chorismate mutase (ScCM) is a typical
example of the AroQ family as it is a homodimer with one tri-helical
active site fold per monomer (Figure 1).1 Each 30 kDa monomer has
an allosteric binding domain at the dimer interface that can
potentially bind either the negative effector tyrosine or the positive
effector tryptophan. The existence of high-resolution crystal
structures of ScCM with positive effector, negative effector, and
transition state analogue bound provides some evidence of the
changes in tertiary structure that allow for regulation.5,6 Evidence for
the residue-level mechanism also exists from dynamic analysis of
the crystal structures.7 We wish to further supplement this data with
experimental evidence for the residue-resolution mechanism behind
allosteric regulation in ScCM through NMR spectroscopy.
The sample conditions for studying ScCM have been developed
and NMR titration studies have identified resonances responsive to
allosteric effects, and suggest structural dynamic changes (Figure
2). Additional work is currently being done to assign backbone and
side chain resonances through traditional methods towards the
goal of understanding the network of residues involved in allostery.
As this project is completed, future work will be aimed towards
studying other AroQ family chorismate mutases such as secreted
Mycobacterium tuberculosis chorismate mutase and comparing
their amino acid networks to the ones present in ScCM.
References and Acknowledgements:
1. Helmstaedt, K., Krappmann, S., Braus, G. Allosteric regulation of catalytic activity: Escherichia coli aspartate
transcarbamoylase versus yeast chorismate mutase. MMBR, 65(3), 303-421 (2001)
2. Haslam, E. Shikimic Acid Metabolism and Metabolites. John Wiley & Sons: New York, 1993.
3. Kim, S., Reddy, S., Nelson, B., Vasquez, G., Davis, A., Howard, A., Patterson, S., Gilliland, G., Ladner, J., Reddy,
P. Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium
tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids. Journal of Bacteriology, 188(24),
8638-8648 (2006)
4. Vanholme, B., Kast, P., Haegeman, A., Jacob, J., Grunewald, W., Gheysen, G. Structural and functional
investigation of a secreted chorismate mutase from the plant-parasitic nematode Heterodera schachtii in the context
of related enzymes from diverse origins. Molecular Plant Pathology, 10(2), 189-200 (2009)
5. Strater, N., Hakansson, K., Schnappauf, G., Braus, G., Lipscomb, W. Crystal structure of the T state of allosteric
yeast chorismate mutase and comparison with the R state. PNAS, 93, 3330-3334 (1996)
6. Strater, N., Schnappauf, G., Braus, G., Lipscomb, W. Mechanisms of catalysis and allosteric regulation of yeast
chorismate mutase from crystal structures. Structure, 5, 1437-1452 (1997)
7. Kong, Y., Ma, J., Karplus, M., Lipscomb, W. The allosteric mechanism of yeast chorismate mutase: A dynamic
analysis. Journal of Molecular Biology, 356(1), 237-247 (2006)
This work was partly funded through NSF Career grant MCB-1053993.