Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect

Journal Article · Thu Jan 05 00:00:00 EST 2012 · Biochemistry

DOI:https://doi.org/10.1021/bi201609n· OSTI ID:1053365

Sarma, Akella V. S.; Anbanandam, Asokan; Kelm, Allek; Mehra-Chaudhary, Ritcha; Wei, Yirui; Qin, Peiwu; Lee, Yingying; Berjanskii, Mark V.; Mick, Jacob A.; Beamer, Lesa J.; Van Doren, Steven R.

Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. ¹³C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing k_cat. Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2–3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster ¹⁵N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. ¹⁵N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1–3, and with a different principal axis of diffusion. Finally, this adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.

• Research Organization: Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1053365
• Journal Information: Biochemistry, Vol. 51, Issue 3; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

Related Subjects

Environmental Molecular Sciences Laboratory