Mapping of the active site zinc ligands of peptide deformylase.
A set of 50 site-directed mutants of the Escherichia coli fms gene was constructed to delineate the residues of the active site of peptide deformylase, including the ligands of the zinc ion. In particular, because zinc is usually coordinate by Asp, Cys, Glu or His residues, all the corresponding codons were individually changed. The functional consequence of the substitutions was assessed by complementation of a fms-null strain with the help of vectors expressing the mutate genes. In addition to the mutations of the Cys90 codon, only those of the three conserved residues of the 132HEXXH136 motif of peptide deformylase prevented the indicator strain growing. Most enzyme variants were purified to homogeneity in a second step. Their characterization in vitro showed that the defects in complementation as observed in vivo corresponded to huge decreases of deformylation efficiency. The change of Glu88 also led to a significant decrease in catalytic rate. Unexpectedly, upon substitutions of Glu79 or of Glu83, the enzymes exhibited a strongly increased catalytic efficiency. The measurement of the content of zinc in each purified variant indicated that Cys90, His132 and His136 bound the metal ion. Zinc-free variants mutated at these positions were obtained and shown to display an increased sensitivity to proteolytic attack. Altogether, the data showed that both the presence of zinc and the conserved residues of the HEXXH motif were crucial for the activity of deformylase. This behaviour identified the enzyme as a member of the zinc metalloproteases superfamily. However, the unexpected participation in the binding of the zinc atom of Cys90, upstream from the HEXXH motif, suggested that peptide deformylase could be representative of a new sub-family, distinct from those of thermolysin and astacin.
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005816:Genetic Complementation Test; D008024:Ligands; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000145:classification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D008024:Ligands; D000626:Aminopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase; D015032:Zinc