Vol. 175, No. 23 Evidence that Peptide Deformylase and Methionyl-tRNAet Formyltransferase Are Encoded within the Same Operon in Escherichia coli THIERRY MEINNEL* AND SYLVAIN BLANQUET Laboratoire de Biochimie, Unite de Recherche Associ'ee, no. 240 du Centre National de la Recherche Scientifique, Ecole Polytechnique, F-91128 Palaiseau Cedex, France Received 19 July 1993/Accepted 30 September 1993 Overexpression of theftns gene, the first translation unit of a dicistronic operon that also encodes methionyl- tRNAet formyltransferase in Escherichia col, sustains the overproduction of peptide deformylase activity in crude extracts. This suggests that thefins gene encodes the peptide deformylase. Moreover, thefins gene product has a motif characteristic of metalloproteases, an activity compatible with deformylase. The corresponding protein could be purified to homogeneity. However, its enzymatic activity could not be retained during the purification procedure. As could be expected from the occurrence in its amino acid sequence of a zinc-binding motif characteristic of metallopeptidases, the purified fins product displayed one tightly bound zinc atom. Translation of mRNAs into proteins most often initiates on a methionine codon. This initiator codon corresponds to a specialized initiator methionine tRNA. In addition, in pro- karyotes as well as in the chloroplasts and mitochondria of eukaryotes, a formyl group is added to the NH2 of the methionine esterified to initiator methionine tRNA prior to the involvement of this tRNA in translation (10, 11, 19, 27) and formylmethionine instead of methionine is incorporated at the N terminus of nascent proteins (2, 5, 8, 31). However, exami- nation of the N-terminal residue of the proteins synthesized in vivo in prokaryotes as well as in organelles reveals that the N-formylmethionine is not retained. Methionine, alanine, and serine were most frequently found as N-terminal residues (7, 15, 19, 30). It was early proposed that the amino-terminal formylmethi- onine of a nascent protein could be removed posttranslation- ally (2, 9). In agreement with this hypothesis, two distinct enzymatic activities could be described: (i) a peptide deformy- lase capable of cleaving the formyl group from formylmethi- onine peptides (1, 18, 28, 32) and (ii) a methionine aminopep- tidase which removes the N-terminal methionine from methionine peptides (1, 18, 23, 28, 32). The gene encoding this methionine aminopeptidase has been identified in Escherichia coli (4). Studies of the specificity of the corresponding protein both in vivo and in vitro indicate that removal of the initiator methionine is favored when the length of the side chain of the second amino acid is short enough (4, 14). In contrast to methionine aminopeptidase, the substrate specificity of pep- tide deformylase appears insensitive to the nature of the second residue following methionine (1). Actually, removal of the N-terminal formyl group from formylmethionine peptides occurs systematically. The coupled specificities of the two enzymes therefore explain why alanine, glycine, serine, or valine on the one hand and methionine on the other are usually found as N-terminal amino acids of proteins. However, peptide deformylase could not be further characterized be- cause of its extreme instability (1, 18, 28). The lability of peptide deformylase activity might be the reason why a formyl- * Corresponding author. Electronic mail address: Labo@coli.poly- technique.fr. methionine group is retained on nascent polypeptides synthe- sized in cell extracts from E. coli (1). The fnt gene encoding the enzyme which catalyzes the formylation of Met-tRNAMet (methionyl-tRNAf et formyl- transferase) has been recently cloned in our laboratory (13). The product of the fint gene plays an essential role for optimal growth of E. coli cells (13), possibly by enabling Met- Met tRNAf to participate in the initiation rather than in the elongation step of the translation process (12). fnt is the second cistron of an operon whose first cistron, which we called fins, encodes a putative 19-kDa polypeptide (12, 21). The translated amino acid sequence of fins shows a motif charac- teristic of neutral zinc aminopeptidases (16, 17, 25, 29) (Fig. 1). Since the enzyme function of E. coli peptide deformylase resembles that of an aminopeptidase, it is possible that the fins gene encodes peptide deformylase. To test this hypothesis, we cloned the fns gene and expressed it from an inducible promoter. Peptide deformylase activity can be overexpressed from a plasmid carrying fins. The 821-bp XbaI-PstI restriction frag- ment of the pBS936XB plasmid (13), which carries onlyfins as a full-length open reading frame, was ligated between the XbaI and PstI restriction sites of pUC18, yielding the pUCdef plasmid in which the fins gene was placed under the control of the Lac promoter. JMlOlTr cells (10 ml) carrying either the pUCdef or the pUC18 control plasmids were grown overnight in the presence of 0.3 mM isopropyl-p-D-galactopyranoside (IPTG) and 50 ,ug of ampicillin per ml. After centrifugation, cells were resuspended in 1 ml of 50 mM HEPES (N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid [pH 7.0]) and sonicated. Cells debris were removed by centrifugation, and the total protein concentration in the cell extracts was mea- sured (6). An aliquot (0.5 ,ul) of each of the two extracts was analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE [20% polyacrylamide gel; Phast-system; Pharmacia]). One protein band with an apparent molecular mass of 23 ± 1 kDa was specifically overexpressed in JMlOlTr (pUCdef) extracts (Fig. 2). This band was absent from the gel with the JMlOlTr (pUC18) cell extracts (Fig. 2). Peptide deformylase activity in each extract was measured with a method similar to that previously described by Adams 7737 JOURNAL OF BACrERIOLOGY, Dec. 1993, p. 7737-7740 0021-9193/93/237737-04$02.00/0 on June 17, 2020 by guest http://jb.asm.org/ Downloaded from