Triple resonance and relaxation dispersion studies of the enzyme phosphomannomutase/phosphoglucomutase
EMSL Project ID
21492
Abstract
The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is a member of the a-D-phosphohexomutase superfamily. These proteins have varied roles in carbohydrate metabolism and other biosynthetic pathways, and at least one representative of the family appears to be present in all organisms from bacteria to humans. PMM/PGM from P. aeruginosa participates in the biosynthesis of several bacterial molecules, including the complex glycolipid lipopolysaccharide, the exopolysaccharide known as alginate, and the surfactant rhamnolipid. The structure of PMM/PGM (and complexes with its four substrates) has been characterized at high resolution by X-ray crystallography: the protein is a monomer (463 residues) with four domains, and a large active site cleft. Crystallographic studies of PMM/PGM have shown that significant domain rotation occurs upon binding to substrates, implying a key role for dynamics in its reaction mechanism. We have therefore embarked on NMR studies of PMM/PGM to address unexplored aspects of its reaction mechanism, including the role of dynamics in catalysis. The reaction of PMM/PGM is highly reversible, and our crystallographic studies suggest that domain rotation is required at multiple times during catalysis (Regni, et al. JBC 2006). We plan to characterize the dynamics of the enzyme during catalysis using relaxation dispersion studies, as described by Kern and coworkers in their 2005 Nature paper. Because of the large size of PMM/PGM, triple resonance studies will first be necessary to assign its spectrum. We have obtained a preliminary TROSY of 15N, 2H PMM/PGM at 0.4 mM, 34° C and pH 7.4, showing that the spectrum is well dispersed and that more than 95% of the potential peaks are present (see attached JPEG image). This data was collected at 800 MHz with cryogenic probe at the Univ. of Kansas NMR facility. We therefore request approximately 2 weeks of time on the 800 MHz magnet with cryoprobe that is available at PNNL. Based on our experience at the Kansas facility, it takes >2 hr. to get a complete TROSY at 0.4 mM, even with perdeuteration. Two weeks of magnet time should allow us to get the most important triple resonance spectra (HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, HNCO, HN(CA)CO) for assigning the spectrum. In addition, we would like to request another 10 days to 2 weeks to obtain 15N SQ dispersion curve from a dozen TROSY-CPMG series, if possible. This will complement the TROSY-CPMG that works well routinely on our home-system Inova 600 w/ cold probe.
Project Details
Project type
Capability Research
Start Date
2007-01-02
End Date
2007-07-18
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Sarma AV, A Anbanandam, A Kelm, R Mehra-Chaudhary, Y Wei, P Qin, Y Lee, MV Berjanskii, JA Mick, LJ Beamer, and SR Van Doren. 2012. "Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect." Biochemistry 51(3):807?819. doi:10.1021/bi201609n