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 nasc ent polypeptides and represents a potential target for drug discovery, was determined using N-15/C-13 doubly labeled protein. Nearly completely automa ted assignment routines were employed to assign three-dimensional triple re sonance, N-15-resolved and C-13-resolved NOESY spectra using the program GA RANT. This assignment strategy, demonstrated on a 17 kDa protein, is a sign ificant advance in the automation of NMR data assignment and structure dete rmination 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 An gstrom for the root-mean-square deviation calculated from the backbone atom s N, C-alpha and C', and 0.81 Angstrom for all heavy atoms of the individua l conformers relative to the mean coordinates for residues 1 to 150. The gl obular fold of PDF contains two cl-helices comprising residues 25-40, 125-1 38, 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 HE XXH motif positioning a zinc ligand in a similar fashion to other metallopr oteases, with the third ligand being cysteine and the fourth presumably a w ater. The three-dimensional structure of PDF affords insight into the subst rate recognition and specificity for N-formylated over N-acetylated substra tes and is compared to other PDF structures.