Dynamics of Bacterial Phosphoglucomutase via NMR Relaxation
EMSL Project ID
40091
Abstract
A bacterial enzyme known as phosphomannomutase / phosphoglucomutase (PMM/PGM) is required in the biosynthesis of the sugar capsule. Bacteria use this coating of complex carbohydrates in forming their environmental communities known as biofilms and in the virulence of pathogenic bacteria. One such pathogen is Pseudomonas aeruginosa found in lungs of cystic fibrosis patients, in burn wounds, and in soil. Thorough characterization of enzyme kinetics and catalytically-linked structural opening and closing of the active site of PMM raise the question of the nature of the motions facilitating this. We used 800 MHz triple resonance NMR to assign over 90% of the backbone amide peaks of wild-type PMM from P. aeruginosa. We are also assigning the amide peaks of inactivated PMM and have obtained at least 55% of them. Spectral crowding in the center of the TROSY spectra remains a limiting problem for quantitative NMR-based dynamics studies of this 51 kDa enzyme of 463 residues. 900 MHz time is requested for investigating the following two related questions: (I) Will binding of a glucose-phosphate analog inhibitor partly quench millisecond-scale motions expected to be intrinsic to the free state of active PMM? (II) How will binding of the glucose-1-phosphate substrate to inactivated PMM modify the millisecond fluctuations intrinsic to its free state? The 900 MHz spectrometer time is needed for its remarkably better resolution of the central region of the TROSY spectrum of PMM which remains too crowded at 800 MHz and for the amplification of the NMR line broadening accompanying structural fluctuations on the millisecond timescale. Addressing each question is estimated to require at least 8 days of 900 MHz time: 4 days for each free state and 4 days for each bound state. Three TROSY-enhanced 15N NMR relaxation series (R2a, R1 2HzNz, nxy) will be run on each state. We report our success in 2009 with such measurements using EMSL 800 MHz, both on the PMM enzyme and with homologous proteolytic enzymes that we compared (Liang et al., 2010, Biophysical J.). Preliminary data suggest that comparison of sites of millisecond motions intrinsic to the free state of PMM with its sites sensitive to shifts of NMR peaks when glucose-phosphate substrates bind may prove quite interesting. Such comparisons were used elegantly by Boehr and Wright to demonstrate that states of the enzyme dihydrofolate reductase sample excited states resembling the next step in the catalytic progression. We will use the 900 MHz relaxation measurements to test whether this idea of an energy landscape progressively shifting to promote catalysis may apply also to the class of enzymes represented by PMM.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2010-10-01
End Date
2012-09-30
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