Structure-function relationships within the peptide deformylase family. Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion.
Thermus thermophilus peptide deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure.
Meinnel
T
T
Unite de Recherche Associee n 1970 du Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
Lazennec
C
C
Villoing
S
S
Blanquet
S
S
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology; D015032:Zinc / Q000737:chemistry*
D000626:Aminopeptidases; D008666:Metalloendopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase; D015032:Zinc