Michaël Delmarcelle

The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP b-lactamase

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP β‐lactamases by β‐lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaR1 genes share a high degree of sequence homology and act as repressors and penicillin‐sensory transducers respectively. It has been shown in S. aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study, we followed the fate of BlaI during β‐lactamase induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blaI and blaR1 genes. In contrast to the situation in B. licheniformis 749/I, β‐lactamase induction in B. subtilis 168/pDML995 was not correlated with the proteolysis of BlaI. To exclude molecular variations undetectable by SDS–PAGE, two‐dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells. No variation in the BlaI isoelectric point was observed in induced cells, whereas the DNA‐binding property was lost. Cross‐linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non‐covalent modification by a co‐activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co‐activator, our results show that the presence of penicillin stress is also required for full induction of β‐lactamase biosynthesis.

Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the 'penicillin-recognizing enzyme' family.

BACKGROUND beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis. The most common bacterial resistance mechanism against beta-lactam compounds is the synthesis of beta-lactamases that hydrolyse beta-lactam rings. These enzymes are believed to have evolved from cell-wall DD-peptidases. Understanding the biochemical and mechanistic features of the beta-lactam targets is crucial because of the increasing number of resistant bacteria. DAP is a D-aminopeptidase produced by Ochrobactrum anthropi. It is inhibited by various beta-lactam compounds and shares approximately 25% sequence identity with the R61 DD-carboxypeptidase and the class C beta-lactamases. RESULTS The crystal structure of DAP has been determined to 1.9 A resolution using the multiple isomorphous replacement (MIR) method. The enzyme folds into three domains, A, B and C. Domain A, which contains conserved catalytic residues, has the classical fold of serine beta-lactamases, whereas domains B and C are both antiparallel eight-stranded beta barrels. A loop of domain C protrudes into the substrate-binding site of the enzyme. CONCLUSIONS Comparison of the biochemical properties and the structure of DAP with PBPs and serine beta-lactamases shows that although the catalytic site of the enzyme is very similar to that of beta-lactamases, its substrate and inhibitor specificity rests on residues of domain C. DAP is a new member of the family of penicillin-recognizing proteins (PRPs) and, at the present time, its enzymatic specificity is clearly unique.

IND-6, a Highly Divergent IND-Type Metallo-β-Lactamase from Chryseobacterium indologenes Strain 597 Isolated in Burkina Faso

ABSTRACT The genus Chryseobacterium and other genera belonging to the family Flavobacteriaceae include organisms that can behave as human pathogens and are known to cause different kinds of infections. Several species of Flavobacteriaceae, including Chryseobacterium indologenes, are naturally resistant to β-lactam antibiotics (including carbapenems), due to the production of a resident metallo-β-lactamase. Although C. indologenes presently constitutes a limited clinical threat, the incidence of infections caused by this organism is increasing in some settings, where isolates that exhibit multidrug resistance phenotypes (including resistance to aminoglycosides and quinolones) have been detected. Here, we report the identification and characterization of a new IND-type variant from a C. indologenes isolate from Burkina Faso that is resistant to β-lactams and aminoglycosides. The levels of sequence identity of the new variant to other IND-type metallo-β-lactamases range between 72 and 90% (for IND-4 and IND-5, respectively). The purified enzyme exhibited N-terminal heterogeneity and a posttranslational modification consisting of the presence of a pyroglutamate residue at the N terminus. IND-6 shows a broad substrate profile, with overall higher turnover rates than IND-5 and higher activities than IND-2 and IND-5 against ceftazidime and cefepime.

Specificity inversion of Ochrobactrum anthropi D‐aminopeptidase to a D,D‐carboxypeptidase with new penicillin binding activity by directed mutagenesis

The serine penicillin‐recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to β‐lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D‐aminopeptidase and of the Streptomyces R61 D,D‐carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D‐aminopeptidase lost its original activity in favor of a new penicillin‐binding activity. D‐aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild‐type enzyme. By a second step site‐directed mutagenesis, the specificity of the Ochrobactrum anthropi D‐aminopeptidase was inverted to a D,D‐carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin‐recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin‐recognizing proteins are discussed.

Structure of the Archaeal Pab87 Peptidase Reveals a Novel Self-Compartmentalizing Protease Family

Self-compartmentalizing proteases orchestrate protein turnover through an original architecture characterized by a central catalytic chamber. Here we report the first structure of an archaeal member of a new self-compartmentalizing protease family forming a cubic-shaped octamer with D 4 symmetry and referred to as CubicO. We solved the structure of the Pyrococcus abyssi Pab87 protein at 2.2 Å resolution using the anomalous signal of the high-phasing-power lanthanide derivative Lu-HPDO3A. A 20 Å wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 Å wide central chamber holding the eight active sites. Surprisingly, activity assays revealed that Pab87 degrades specifically d-amino acid containing peptides, which have never been observed in archaea. Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism. We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins.

A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

ABSTRACT We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase.

The specificity of the Streptomyces R61 penicillin‐sensitive D‐Ala‐D‐Ala peptidase has been re‐examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly‐L‐Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D‐Ala‐D‐Ala peptidases. The Gly‐L‐Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly‐L‐Xaa peptide or D‐Alanine are supplied as acceptors. Utilization of substrates in which the terminal ‐COO− group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibilty of the transpeptidation reaction.

Synthesis and Physicochemical Characterization of D -Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D -Tagatose Catabolic Pathway of Bacillus licheniformis

We report the first enzymatic synthesis of <smlcap>D</smlcap>-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by ³¹P and ¹H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated <smlcap>D</smlcap>-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF^His6) of Escherichia coli. The active fusion enzyme was named TagK-TF^His6. Tag-1P and <smlcap>D</smlcap>-fructose-1-phosphate are substrates for the TagK-TF^His6 enzyme, whereas the isomeric derivatives <smlcap>D</smlcap>-tagatose-6-phosphate and <smlcap>D</smlcap>-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (k_cat/K_m) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.