Colette Goffin

Lack of Cell Wall Peptidoglycan versus Penicillin Sensitivity: New Insights into the Chlamydial Anomaly

Intracellular bacterial pathogens enter their hosts surrounded by a membrane-bound vacuole and use a panel of tricks to exploit or evade eukaryotic cell functions ([9][1],[12][2]). Chlamydia inhabits vesicles that do not fuse with lysosomes and remains within these parasitophorous vacuoles (termed

The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family

The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

The dppA gene of Bacillus subtilis encodes a new d‐aminopeptidase

Different strains of Bacillus were screened for their ability to hydrolyse d‐alanyl‐p‐nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus subtilis 168. The last strain was the best producer and was selected for the production and purification of the enzyme. The determination of the N‐terminal sequence identified the enzyme as the product of the dppA gene (previously named dciAA) belonging to the dipeptide ABC transport (dpp) operon expressed early during sporulation. Open reading frames (ORFs) encoding putative related proteins were found in the genomes of a variety of Archaea and both sporulating and non‐sporulating bacteria. The enzyme behaves as a d‐aminopeptidase and represents the prototype of a new peptidase family. Among the tested substrates, the highest activities were found with d‐Ala‐d‐Ala and d‐Ala‐Gly‐Gly. The active enzyme behaves as an octamer of identical 30 kDa subunits. It exhibits a broad pH optimum, extending between pH 9 and 11. It is reversibly inhibited in the presence of Zn2+ chelators, and the sequence comparisons highlight the conservation of potential Zn‐binding residues. As it has been shown by others that null mutations in the dpp operon do not inhibit spore formation, the physiological role of DppA is probably an adaptation to nutrient deficiency.

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide

Abstract. Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172.

Some properties of the interstrand crosslinks in depurinated DNA

The interstrand crosslinks that appear in stored depurinated DNA interfere with the counting of apurinic sites and strand breaks by sucrose gradient analysis. They could not be cleaved at acid or alkaline pH, or by treatment with methoxyamine.

Interstrand DNA crosslinks due to AP (apurinic/apyrimidinic) sites

Storage of a solution of DNA containing apurinic sites, even at 4°C leads to the appearance of interstrand crosslinks. Possible biological consequences of these crosslinks, when they appear in cell DNA, are briefly discussed. Formation of interstrand crosslinks in DNA containing tritium‐labelled thymine and kept in an aqueous solution might be due, at least partly, to the loss of bases by the autoirradiated DNA.

d-Aminopeptidase DppA

Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of D-Aminopeptidase DppA. The DppA D-aminopeptidase of Bacillus subtilis 168 was discovered by screening different strains of Bacilli for their ability to hydrolyze D-alanyl-p-nitroanilide (DAla-NHPhNO 2 ). The best production was obtained after cell lysis of aging cultures grown in an optimized medium. The determination of the N-terminal sequence of the purified enzyme identified it as the product of the dppA gene (previously named dciAA), the first ORF of the dipeptide permease (dpp) operon expressed early during sporulation. The crystal structure of B. subtilis DppA reveals an elaborate quaternary organization. The general architecture of the enzyme complex is similar to that found in ‘self-compartmentalizing’ proteases, such as the proteasomes and ClpP endopeptidase complexes. Monomeric B. subtilis DppA (274 amino acid residues) consists of two separate domains. The N-terminal domain (residues 1–119 and 132–213) is largely responsible for the construction of the active site. The DppA D-aminopeptidase of B. subtilis shares 76% identity with the product encoded by the dppA gene of B. methanolicus.