STRUCTURE-FUNCTION-RELATIONSHIPS WITHIN THE PEPTIDE DEFORMYLASE FAMILY - EVIDENCE FOR A CONSERVED ARCHITECTURE OF THE ACTIVE-SITE INVOLVING3 CONSERVED MOTIFS AND A METAL-ION

Thermus thermophilus peptide deformylase was characterized. Its enzyma tic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite f ew sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly c onserved amino acids was noticed, EGCLS the cysteine of which correspo nds to the third zinc ligand. The study of site-directed mutants of th e E. coli deformylase shows that the residues of this stretch are cruc ial for the structure and/or catalytic efficiency of the active enzyme . Both aforementioned sequences were used as markers of the peptide de formylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearotherm ophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtil us and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confi rmed that it actually corresponded to a peptide deformylase with prope rties similar to those of the E. coli enzyme. Alignment of the nine pe ptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This mot if is also located in the active site according to the three-dimension al structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residu es for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of pep tide deformylases build up the active site around a metal ion. Finally , an analysis of the location of the other conserved residues, in part icular of the hydrophobic ones, was performed using the three-dimensio nal model of the E. coli enzyme. This enables us to suggest that all b acterial peptide deformylases adopt a constant overall tertiary struct ure. (C) 1997 Academic Press Limited.