STRUCTURE OF PEPTIDE DEFORMYLASE AND IDENTIFICATION OF THE SUBSTRATE BINDING SITE
Andreas Becker1, Ilme Schlichting2, Wolfgang Kabsch1, Sabine Schultz3, A. F. Volker Wagner3
1Max-Planck-Institut für medizinische
Forschung, Abteilung Biophysik, Jahnstrasse 29, 69120 Heidelberg,
Germany
e-mail: abecker@mpimf-heidelberg.mpg.de
2Max-Planck-Institut für molekulare
Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany
3Biochemie-Zentrum Heidelberg,
Ruprecht-Karls Universität Im Neuenheimer Feld 501, 69120
Heidelberg, Germany \vskip
In eubacteria as well as in mitochondria and chloroplasts the amino group of methionyl-tRNAMetf is N-formylated by a formyltransferase during initiation of protein synthesis. Consequently, all nascent polypeptides are synthesized with N-formyl-methionine at the N-terminus. During elongation of the polypeptide chain the formyl group is removed hydrolytically by the enzyme peptide deformylase (EC 3.5.1.27). This formylation/deformylation cycle, which appears to be a characteristic feature of eubacteria, does not occur in the cytoplasm of eucaryotic cells. Therefore, PDF is an attractive target for the design of new antibiotics.
PDF from E. coli, a monomeric protein of 168 residues, employs a Fe2+-ion as catalytic metal [1], [2] and nearly retains its activity on substitution by Ni2+ [1] whereas the Zn2+ form of the enzyme proved virtually inactive [1], [2]. The Zn2+ form of PDF was solved by NMR [3] and x-ray crystallography [4]. Recently, we have described the catalytically active enzyme in the Ni2+ bound form at 2.5 A resolution, and at 1.9 A resolution in complex with a polyethylene glycol molecule, a competitive inhibitor of the enzyme [5]. The Ni2+-ion is tetrahedrally coordinated by Cys 90, His 132, His 136, and a well-defined water molecule. The PEG inhibitor is found close to the metal, earmarking the active site of the enzyme which involves nearly all conserved amino acid residues. Despite the presence of the sequence motif H132 EXXH, known to be involved in zinc binding, the overall arrangement of secondary and tertiary structure is quite different from other metalloproteases such as thermolysin. Detailed studies of crystals of the enzyme in the active nickel and iron-forms as well as in the inactive zinc-form in the presence and absence of substrate/products and inhibitors are underway [6].