Dynamic characterization of the enzyme phosphomannomutase/phosphoglucomutase from P. aeruginosa
EMSL Project ID
21492a
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). Our long term goal is to characterize the dynamics of the enzyme during catalysis using relaxation dispersion studies, as described by Kern and coworkers in their 2005 Nature paper. Our initial step in this process was to collect assignment of ligand-free apo-PMM/PGM and preliminary relaxation dispersion spectra. In this proposal, we request 800 MHz time to characterize the dynamics of PMM/PGM in the presence of two ligands. Based on our previous experience, two weeks of 800 MHz time per liganded state with cryo-probe will allow us to collect the most important triple resonance spectra (HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, HNCO, HN(CA)CO) for that state. In addition, we would like to request another 7 to 10 days per ligand complex to obtain a 15N SQ dispersion curve from using a TROSY-CPMG sequence and, if time permits, a NOESY-TROSY. Each additional state with inhibitor or active catalysis should require similar amounts of 800 time.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-11-01
End Date
2009-09-30
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