CHARACTERIZATION OF NADP(- BACKBONE ATOM NMR ASSIGNMENTS AND CHEMICAL-SHIFT CHANGES() BINDING TO PERDEUTERATED MURB )

Backbone-atom resonances have been assigned for both the substrate-fre e and the NADP(+)-complexed forms of UDP-N-acetylenolpyruvylglucosamin e reductase (MurB), a monomeric, 347-residue (38.5 kDa) flavoenzyme es sential for bacterial cell-wall biosynthesis. NMR studies were perform ed using perdeuterated, uniformly C-13/N-15-labeled samples of MurB. L n the case of substrate-free MurB, one or more backbone atoms have bee n 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-1 3(alpha) atoms (95%), and 297 C-13(beta) atoms (93%). For NADP(+)-comp lexed MurB, one or more backbone atoms have been assigned for 313 resi dues (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) atom s (84%). The strategies used for obtaining resonance assignments are d escribed 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. Th ese data are compared to X-ray crystallographic structures of substrat e-free MurB and MurB complexed with the UDP-N-acetylglucosamine enolpy ruvate (UNAGEP) substrate. NADP(+) binding induces significant chemica l-shift changes in residues both within the known UNAGEP and FAD bindi ng pockets and within regions known to undergo conformational changes upon UNAGEP binding. The NMR data indicate that NADP(+) and UNAGEP uti lize the same binding pocket and, furthermore, that the binding of NAD P(+) 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 reson ances. Overall, our results are consistent with MurB having a flexible active site. (C) 1997 Academic Press Limited.