A high quality nuclear magnetic resonance solution structure of peptide deformylase from Escherichia coli: application of an automated assignment strategy using GARANT

• Published in: Journal of biomolecular NMR 1999-04, Vol.13 (4), p.311-324
• Main Authors: O'Connell, J F, Pryor, K D, Grant, S K, Leiting, B
• Format: Article
• Language: English
• Subjects: Amidohydrolases; Amino Acid Sequence; Aminopeptidases - chemistry; Aminopeptidases - genetics; Automation; Carbon Isotopes; Conserved Sequence; E coli; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Models, Molecular; Molecular Sequence Data; Nitrogen Isotopes; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; Recombinant Proteins - chemistry; Software; Solutions; Thermodynamics
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1023/A:1008311502626

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.