No abstract

The Escherichia coli dapF gene was isolated from a cosmid library as a result of screening for clones overproducing diaminopimelate epimerase. Insertional mutagenesis was performed on the cloned dapF gene with a mini-Mu transposon, leading to chloramphenicol resistance. One of these insertions was transferred onto the chromosome by a double-recombination event, allowing us to obtain a dapF mutant. This mutant accumulated large amounts of LL-diaminopimelate, confirming the blockage in the step catalyzed by the dapF product, but did not require meso-diaminopimelate for growth. The dapF gene was localized in the 85-min region of the E. coli chromosome between cya and uvrD. meso-Diaminopimelate (meso-DAP) is the direct precursor of lysine and is an essential component of the cell wall peptidoglycan in gram-negative bacteria. The formation of this key intermediate is catalyzed by DAP epimerase, an enzyme found several years ago in Escherichia coli (2) but only recently purified and studied for its catalytic properties (34). Furthermore, this enzyme has been specifically studied as a target for antibacterial effects (15).As a step toward an understanding of the regulatory pattern of the whole lysine-DAP biosynthesis pathway in E. coli, we have begun to study the structure and expression of the gene for DAP epimerase (dapF, following the nomenclature of Bukhari and Taylor [6]). No mutants blocked in this enzymic step are available for gene cloning experiments. Consequently, we followed the method of Mechulam et al. (22), screening an E. coli cosmid library and assuming that a strain harboring a multicopy plasmid carrying the dapF gene would overproduce DAP epimerase. The gene was thus cloned and further localized by insertional mutagenesis. A mutation in the cloned gene allowed us to obtain for the first time a chromosomal dapF mutant. This mutation was then used for genetic mapping of the dapF locus on the E. coli chromosome. MATERIALS AND METHODSE. coli strains and plasmids used in this study are listed in Table 1. General genetic and cloning techniques have been described previously (11,25). DAP epimerase assay. DAP epimerase was assayed by the epimerase-catalyzed release of 3H to water from [G-3H]DAP after a 40-min incubation at 25°C as described previously (34). Typically, 100 ,ul of a reaction mixture containing 0.1 M Tris hydrochloride (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol, and 0.5 p.Ci of (DL plus meso)-2,6-diamino[G-3H]pimelic acid dihydrochloride (Radiochemical Centre, Amersham, U.K.) (1 Ci/mmol) was acidified with 500 p1l of 10% trichloroacetic acid and applied to a column (1 ml) of Bio-Rad AG5OW-X 4 ion-exchange resin (H+ form). The column was washed three times with 500 RI of water, and the eluates were combined and counted for radioactivity. Protein was * Corresponding author. measured by the Bradford procedure (5). Specific activities are expressed as counts per minute of 3H20 liberated from DAP per milligram of protein per minute.Screening of the cosmid library. A total of 450 clones from the E. coli...

The dapE gene of Escherichia coli encodes N-succinyl-L-diaminopimelic acid desuccinylase, an enzyme that catalyzes the synthesis of LL-diaminopimelic acid, one of the last steps in the diaminopimelic acid-lysine pathway. The dapE gene region was previously purified from a lambda bacteriophage transducing the neighboring purC gene (J. Parker, J. Bacteriol. 157:712-717, 1984). Various subcloning steps led to the identification of a 2.3-kb fragment that complemented several dapE mutants and allowed more than 400-fold overexpression of N-succinyl-L-diaminopimelic acid desuccinylase. Sequencing of this fragment revealed the presence of two closely linked open reading frames. The second one encodes a 375-residue, 41,129-M(r) polypeptide that was identified as N-succinyl-L-diaminopimelic acid desuccinylase. The first one encodes a 118-residue polypeptide that is not required for diaminopimelic acid biosynthesis, as judged by the wild-type phenotype of a strain in which this gene was disrupted. Expression of the dapE gene was studied by monitoring amylomaltase activity in strains in which the malPQ operon was under the control of various fragments located upstream of the dapE gene. The major promoter governing dapE transcription was found to be located in the adjacent orf118 gene, while a minor promoter allowed the transcription of both orf118 and dapE. Neither of these two promoters is regulated by the lysine concentration in the growth medium.

Recombinant diaminopimelate epimerase is overproduced to give 1 YO of soluble protein when grown under the appropriate conditions in Escherichia coli. This compares with 0.02% of the constitutive level of wild-type enzyme. A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg). This has enabled sufficient amounts of enzyme both to compare with wild-type enzyme and to enable active site modification studies to be performed. Incubation of the enzyme with 2-(4-amino-4-carboxybutyl)-2-aziridine-carboxylic acid (AZIDAP), results in time-dependent irreversible inhibition. Tryptic digestion of the inactivated enzyme and peptide-mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide. Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequencc of the dapF gene shows that Cys73 is labelled. Finally based on limited sequence similarities around this cysteine and active-site cysteines of proline racemase and 1-hydroxyproline 2-epimerase, together with mechanistic considerations, we propose that all three non-pyridoxalphosphate-containing racemases/epimerases derive from a common evolutionary origin.The peptidoglycan layer of the bacterial cell wall is essential to protect against osmotic shock and lysis. Several unusual amino acids, including D-alaninc and n,L-diaminopimelic acid (A,pm), are incorporated into UDP-N-acetylmuramylpentapeptide, the cytoplasmic precursor to peptidoglycan [l]. These unusual amino acids are unique to prokaryotes and should therefore be good targets for the rational design of new antibacterial agents. u-Alanine is formed from L-alanine by alanine racemase and this enzyme is both the target of known antibactcrial agents and the object of extensive studies for the design of new ones [2-51. Although apriorian attractive target, the enzyme diaminopimelate epimerase, which converts L,L-A,pm into L,u-A,pm or meso A2pm, has been little studied, mainly because it is present in low abundance [6]. The enzyme is also interesting per se bccause it is a non-pyridoxalphosphate-containing enzyme and kinetic studies [6] have suggested that it operates via a two-base mechanism analogous to proline racemase [7].Only recently has the location of the dapF gene, which codes for Azpm epimerase, been determined [8]. We isolated the gene and expressed it from a high-copy-number plasmid. Thus we are now able to produce large amounts of the enzyme, making further work possible. Recombinant Azpm epimerase is essentially equivalent to enzyme previously isolated from E. coli strain W [6] (see Results). In addition, we show that Azpm epimerase is irreversibly inactivated by AZIDAP (F. Cierhart et al., unpublished work), a ncw spccific inhibitor of the enzyme that will be described in full elsewhere. We also Ahhreviaiions. AZIDAP, 2-(4-amino-4-carboxybutyl)-2-aziridine carboxylic acid ; A2pm. 2,6-diamiiiopiinelic acid; Cin, carboxymcthyl ; Pth, phcnylthiohydanto...