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 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.
Constantine
K L
KL
Division of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.
Mueller
L
L
Goldfarb
V
V
Wittekind
M
M
Metzler
W J
WJ
Yanchunas
J
J
Robertson
J G
JG
Malley
M F
MF
Friedrichs
M S
MS
Farmer
B T
BT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D002247:Carbon Isotopes; D003903:Deuterium; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D009249:NADP; D003903:Deuterium; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase