Characterization of NADP(+) binding to perdeuterated MurB: Backbone atom NMR assignments and chemical-shift changes

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 C-13/N-15-labeled samples of MurB. Ln the case of substrate-free MurB, one or more backbone atoms have been assigned for 334 residues (96%). The assigned backbone atoms include 309 (HN)-H-1 and N-15 atoms (94%), 315 (CO)-C-13 atoms (91%), 331 C-13(alpha) atoms (95%), and 297 C-13(beta) atoms (93%). For NADP(+)-complexed MurB, one or more backbone atoms have been assigned for 313 residues (90%); these include 283 (HN)-H-1 and N-15 atoms (86%), 305 (CO)-C-13 atoms (88%), 310 C-13(alpha) atoms (89%), and 269 C-13(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 C-13(alpha) and C-13(beta) chemical-shift deviations from random-coil values and from (HN)-H-1-(HN)-H-1 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. (C) 1997 Academic Press Limited.