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Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes.
J Mol Biol. 1997 Apr 18; 267(5):1223-46.JM

Abstract

Backbone-atom resonances have been assigned for both the substrate-free and the NADP+-complexed forms of UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a monomeric, 347-residue (38.5 kDa) flavoenzyme essential for bacterial cell-wall biosynthesis. NMR studies were performed using perdeuterated, uniformly 13C/15N-labeled samples of MurB. In the case of substrate-free MurB, one or more backbone atoms have been assigned for 334 residues (96%). The assigned backbone atoms include 309 1HN and 15N atoms (94%), 315 13CO atoms (91%), 331 13C(alpha) atoms (95%), and 297 13C(beta) atoms (93%). For NADP+-complexed MurB, one or more backbone atoms have been assigned for 313 residues (90%); these include 283 1HN and 15N atoms (86%), 305 13CO atoms (88%), 310 13C(alpha) atoms (89%), and 269 13C(beta) atoms (84%). The strategies used for obtaining resonance assignments are described in detail. Information on the secondary structure in solution for both the substrate-free and NADP+-complexed forms of the enzyme has been derived both from 13C(alpha) and 13C(beta) chemical-shift deviations from random-coil values and from 1HN-1HN NOEs. These data are compared to X-ray crystallographic structures of substrate-free MurB and MurB complexed with the UDP-N-acetylglucosamine enolpyruvate (UNAGEP) substrate. NADP+ binding induces significant chemical-shift changes in residues both within the known UNAGEP and FAD binding pockets and within regions known to undergo conformational changes upon UNAGEP binding. The NMR data indicate that NADP+ and UNAGEP utilize the same binding pocket and, furthermore, that the binding of NADP+ induces structural changes in MurB. Finally, many of the residues within the UNAGEP/NADP+ binding pocket were difficult to assign due to dynamic processes which weaken and/or broaden the respective resonances. Overall, our results are consistent with MurB having a flexible active site.

Authors+Show Affiliations

Division of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article

Language

eng

PubMed ID

9150408

Citation

Constantine, K L., et al. "Characterization of NADP+ Binding to Perdeuterated MurB: Backbone Atom NMR Assignments and Chemical-shift Changes." Journal of Molecular Biology, vol. 267, no. 5, 1997, pp. 1223-46.
Constantine KL, Mueller L, Goldfarb V, et al. Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes. J Mol Biol. 1997;267(5):1223-46.
Constantine, K. L., Mueller, L., Goldfarb, V., Wittekind, M., Metzler, W. J., Yanchunas, J., Robertson, J. G., Malley, M. F., Friedrichs, M. S., & Farmer, B. T. (1997). Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes. Journal of Molecular Biology, 267(5), 1223-46.
Constantine KL, et al. Characterization of NADP+ Binding to Perdeuterated MurB: Backbone Atom NMR Assignments and Chemical-shift Changes. J Mol Biol. 1997 Apr 18;267(5):1223-46. PubMed PMID: 9150408.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes. AU - Constantine,K L, AU - Mueller,L, AU - Goldfarb,V, AU - Wittekind,M, AU - Metzler,W J, AU - Yanchunas,J,Jr AU - Robertson,J G, AU - Malley,M F, AU - Friedrichs,M S, AU - Farmer,B T,2nd PY - 1997/4/18/pubmed PY - 2001/3/28/medline PY - 1997/4/18/entrez SP - 1223 EP - 46 JF - Journal of molecular biology JO - J Mol Biol VL - 267 IS - 5 N2 - Backbone-atom resonances have been assigned for both the substrate-free and the NADP+-complexed forms of UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a monomeric, 347-residue (38.5 kDa) flavoenzyme essential for bacterial cell-wall biosynthesis. NMR studies were performed using perdeuterated, uniformly 13C/15N-labeled samples of MurB. In the case of substrate-free MurB, one or more backbone atoms have been assigned for 334 residues (96%). The assigned backbone atoms include 309 1HN and 15N atoms (94%), 315 13CO atoms (91%), 331 13C(alpha) atoms (95%), and 297 13C(beta) atoms (93%). For NADP+-complexed MurB, one or more backbone atoms have been assigned for 313 residues (90%); these include 283 1HN and 15N atoms (86%), 305 13CO atoms (88%), 310 13C(alpha) atoms (89%), and 269 13C(beta) atoms (84%). The strategies used for obtaining resonance assignments are described in detail. Information on the secondary structure in solution for both the substrate-free and NADP+-complexed forms of the enzyme has been derived both from 13C(alpha) and 13C(beta) chemical-shift deviations from random-coil values and from 1HN-1HN NOEs. These data are compared to X-ray crystallographic structures of substrate-free MurB and MurB complexed with the UDP-N-acetylglucosamine enolpyruvate (UNAGEP) substrate. NADP+ binding induces significant chemical-shift changes in residues both within the known UNAGEP and FAD binding pockets and within regions known to undergo conformational changes upon UNAGEP binding. The NMR data indicate that NADP+ and UNAGEP utilize the same binding pocket and, furthermore, that the binding of NADP+ induces structural changes in MurB. Finally, many of the residues within the UNAGEP/NADP+ binding pocket were difficult to assign due to dynamic processes which weaken and/or broaden the respective resonances. Overall, our results are consistent with MurB having a flexible active site. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/9150408/Characterization_of_NADP+_binding_to_perdeuterated_MurB:_backbone_atom_NMR_assignments_and_chemical_shift_changes_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(97)90915-3 DB - PRIME DP - Unbound Medicine ER -