A high quality nuclear magnetic resonance solution structure of peptide deformylase from Escherichia coli: application of an automated assignment strategy using GARANT.
The NMR structure of the peptide deformylase (PDF) (1-150) from Escherichia coli, which is an essential enzyme that removes the formyl group from nascent polypeptides and represents a potential target for drug discovery, was determined using 15N/13C doubly labeled protein. Nearly completely automated assignment routines were employed to assign three-dimensional triple resonance, 15N-resolved and 13C-resolved NOESY spectra using the program GARANT. This assignment strategy, demonstrated on a 17 kDa protein, is a significant advance in the automation of NMR data assignment and structure determination that will accelerate future work. A total of 2302 conformational constraints were collected as input for the distance geometry program DYANA. After restrained energy minimization with the program X-PLOR the 20 best conformers characterize a high quality structure with an average of 0.43 A for the root-mean-square deviation calculated from the backbone atoms N, C alpha and C', and 0.81 A for all heavy atoms of the individual conformers relative to the mean coordinates for residues 1 to 150. The globular fold of PDF contains two alpha-helices comprising residues 25-40, 125-138, six beta-strands 57-60, 70-77, 85-88, 98-101, 105-111, 117-123 and one 3(10) helix comprising residues 49-51. The C-terminal helix contains the HEXXH motif positioning a zinc ligand in a similar fashion to other metalloproteases, with the third ligand being cysteine and the fourth presumably a water. The three-dimensional structure of PDF affords insight into the substrate recognition and specificity for N-formylated over N-acetylated substrates and is compared to other PDF structures.
O'Connell
J F
JF
Department of Biochemistry, Merck Research Laboratories, Rahway, NJ 07065, USA. oconnell@merck.com
Pryor
K D
KD
Grant
S K
SK
Leiting
B
B
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics; D001331:Automation; D002247:Carbon Isotopes; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012984:Software; D012996:Solutions; D013816:Thermodynamics
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D012996:Solutions; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase