SOLUTION STRUCTURE OF NICKEL-PEPTIDE DEFORMYLASE

In the accompanying paper, we report that zinc is unlikely to be the c ofactor supporting peptide deformylase activity in vivo. Ln contrast, nickel binding promotes full enzyme activity. The three-dimensional st ructure of the resulting nickel-containing peptide deformylase (cataly tic domain, residues 1 to 147) was solved by NMR using a C-13-N-15-dou bly labelled protein sample. A set of 2261 restraints could be collect ed, with an average of 15.4 per amino acid. The resolution, which show s a good definition for the position of most side-chains, is greatly i mproved compared to that previously reported for the zinc-containing, inactive form. A comparison of the two structures indicates however th at both share the same 3D organization. This shows that the nature of the bound metal is the primary determinant of the hydrolytic activity of this enzyme. Site-directed mutagenesis enabled us to determine the conserved residues of PDF involved in the structure of the active site . in particular, a buried arginine appears to be critical for the posi tioning of Cys90, one of the metal ligands. Furthermore, the 3D struct ure of peptide deformylase was compared to thermolysin and metzincins. Although the structural folds are very different, they all display a common structural motif involving an alpha-helix and a three-stranded beta-sheet. These conserved structural elements build a common scaffol d which includes the active site, suggesting a common hydrolytic mecha nism for these proteases. Finally, an invariant glycine shared by both PDF and metzincins enables us to extend the conserved motif from HEXX H to HEXXHXXG. (C) 1998 Academic Press.