C. Damblon - Academia.edu (original) (raw)

Papers by C. Damblon

Research paper thumbnail of Synthesis and biological evaluation of potential positron emission tomography (PET) ligands for brain visualization of dopamine D3 receptors

Arkivoc, 2006

Currently, the lack of a selective dopamine D 3 PET (positron emission tomography) radioligand fo... more Currently, the lack of a selective dopamine D 3 PET (positron emission tomography) radioligand for in vivo brain occupancy studies is problematic. Several requirements are necessary for a potential PET radioligand for visualization of a receptor into the brain: i) high affinity and selectivity for the target receptor; ii) suitable lipophilicity for both blood-brain barrier permeation and low nonspecific binding to proteins and lipids; iii) structural features that allow labelling with a positron emission isotope. In this study the synthesis and binding affinities for dopamine D 3 and D 2 receptors of several N-[4-(4-arylpiperazin-1-yl)butyl]arylcarboxamides are reported. These compounds were designed by the structural modification of the formerly reported D 3 receptor ligand N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-7-methoxy-2benzofurancarboxamide (1), with the aim to obtain a suitable lipophilicity and the structural features for labelling. In particular, both the 2,3-dichlorophenyl group and the 7-methoxy-2benzofurancarboxamide moiety were replaced by less lipophilic fragments. Among the studied compounds, derivatives N-[4-[4-(5-methoxy-2-benzisoxazolyl)piperazin-1-yl]butyl]-4-(4morpholinyl)benzamide (20), N-[4-[4-(5-methoxy-2-benzisoxazolyl)piperazin-1-yl]butyl]-4-(1Himidazol-1-yl)benzamide (21), and N-[4-[4-(5-methoxy-2-benzisoxazolyl)piperazin-1-yl]butyl]-5-(2-furanyl)-1H-pyrazole-3-carboxamide (22) displayed good D 3 receptor affinities (K i values 38, 22.6, and 21.3 nM, respectively) and were found to be inactive at D 2 receptor. Moreover, on the basis of their experimental log P values and their ability to cross the Caco-2 monolayer, compounds 20-22 are likely to permeate the blood-brain barrier, differently from compound 1.

Research paper thumbnail of G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets

Antimicrobial agents and chemotherapy, 2018

G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic a... more G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite , which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has...

Research paper thumbnail of Reinforced poly(hydroxyurethane) thermosets as high performance adhesives for aluminum substrates

Polymer Chemistry, 2017

High performance adhesives for bare aluminum are prepared by reinforcing poly(hydroxyurethane) (P... more High performance adhesives for bare aluminum are prepared by reinforcing poly(hydroxyurethane) (PHU) thermosets with (functional) nanofillers and poly(dimethylsiloxane).

Research paper thumbnail of Identification and characterization of EYK1, a key gene for erythritol catabolism in Yarrowia lipolytica

Applied Microbiology and Biotechnology, 2017

Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipo... more Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipolytica, in response to osmotic stress. This metabolite has application as food additive due to its sweetening properties. Although Y. lipolytica can produce erythritol at a high level from glycerol, it is also able to consume it as carbon source. This ability negatively affects erythritol productivity and represents a serious drawback for the development of an efficient erythritol production process. In this study, we have isolated by insertion mutagenesis a Y. lipolytica mutant unable to grow on erythritol. Genomic characterization of the latter highlighted that the mutant phenotype is directly related to the disruption of the YALI0F01606g gene. Several experimental evidences suggested that the identified gene, renamed EYK1, encodes an erythrulose kinase. The mutant strain showed an enhanced capacity to produce erythritol as compared to the wild-type strain. Moreover, in specific experimental conditions, it is also able to convert erythritol to erythrulose, another compound of biotechnological interest.

Research paper thumbnail of CG7054 solution structure

Research paper thumbnail of Breakdown of the stereospecificity of dd-peptidases and β-lactamases with thiolester substrates

Biochemical Journal, 1995

With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglyc... more With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an ...

Research paper thumbnail of Streptomyces K15 active-site serine dd-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions

Biochemical Journal, 1995

The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacte... more The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of carbonyl donors Z-R1-CONH-CHR2-COX-CHR3-COO- (where X is NH, S or O) and transfers the electrophilic group Z-R1-CONH-CHR2-CO to amino acceptors via an acyl-enzyme intermediate. Kinetic data suggest that the amino acceptor behaves as a simple alternative nucleophile at the level of the acyl-enzyme in the case of thiol ester and ester donors, and that it binds to the enzyme.carbonyl donor Michaelis complex and influences the rate of enzyme acylation by the carbonyl donor in the case of amide donors. Depending on the nature of the scissile bond, the enzyme has different requirements for substituents at positions R1, R2 and R3.

Research paper thumbnail of Direct n.m.r. evidence for substrate-induced conformational changes in a β -lactamase

Biochemical Journal, 1994

Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very... more Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum. The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r. experiments.

Research paper thumbnail of The mechanism of action of dd-peptidases: the role of tyrosine-159 in the Streptomyces R61 dd-peptidase

Biochemical Journal, 1993

Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or ... more Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding was also drastically decreased, except for the reactions with nitrocefin and methicillin. The consequences of the first mutation were more surprising, since a large proportion of the thiolesterase activity was retained, together with the penicillin-binding capacity. Conversely, the peptidase properties was severely affected. In both cases, a drastic decrease in the transferase activity was observed. The results are compared with those obtained by mutation of the corresponding residue in the class A beta-lactamase of Streptomyces albus G.

Research paper thumbnail of Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins

Biochemical Journal, 1991

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membra... more The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.

Research paper thumbnail of Chromogenic depsipeptide substrates for β-lactamases and penicillin-sensitive dd-peptidases

Biochemical Journal, 1990

Various ester and thioester derivatives of hippuric acid have been prepared which were substrates... more Various ester and thioester derivatives of hippuric acid have been prepared which were substrates of both beta-lactamases and DD-peptidases. The thioesters were more rapidly hydrolysed by nearly all the enzymes. Surprisingly, the enzymes acted rather efficiently on substrates which did not contain any chiral centre.

Research paper thumbnail of Complete ¹H, ¹⁵N, and ¹³C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid

Biomolecular NMR assignments, 2014

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and ... more β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96%) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.

Research paper thumbnail of High-Level Biosynthesis of the Anteiso-C₁₇ Isoform of the Antibiotic Mycosubtilin in Bacillus subtilis and Characterization of Its Candidacidal Activity

Research paper thumbnail of Thiolester substrates of dd-peptidases and ?-lactamases

Letters in Peptide Science, 1995

With peptide substrates, the penicillin-sensitive DD-peptidases exhibit a strict specificity for ... more With peptide substrates, the penicillin-sensitive DD-peptidases exhibit a strict specificity for D-AIa-D-Xaa Ctermini. Only glycine is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes also hydrolyse various ester and thiolester analogues of their natural substrates. Some of the thiolesters whose C-terminal leaving group exhibited an L stereochemistry were significantly hydrolysed by some of the studied enzymes, particularly by the Actinomadura R39 DD-peptidase. By contrast, the strict specificity for a D residue in the penultimate position was fully retained. The same esters and thiolesters also behaved as substrates for [3lactamases. In this case, thiolesters exhibiting L stereochemistry in the C-terminal position could also be hydrolysed, mainly by the class C and class D enzymes. But, more surprisingly, the class C Enterobacter cloacae P99 [Mactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes more efficiently than the D isomer.

Research paper thumbnail of Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

PLoS ONE, 2012

Background: Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase in... more Background: Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings: In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K D for different ligands. Native mass spectrometry was used as an alternative method for measuring K D values. Conclusions/Significance: Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20uC to pH 7.5/NaCl 100 mM/30uC, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

Research paper thumbnail of Structural basis for the broad-spectrum inhibition of metallo-β-lactamases by thiols

Organic & Biomolecular Chemistry, 2008

The development of broad-spectrum metallo-b-lactamase (MBL) inhibitors is challenging due to stru... more The development of broad-spectrum metallo-b-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.

Research paper thumbnail of DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase

Nucleic Acids Research, 2000

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of geno... more The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O 6alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O 6-methylguanine (O 6 meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.

Research paper thumbnail of A minimalistic approach to identify substrate binding features in B1 Metallo-β-lactamases

Bioorganic & Medicinal Chemistry Letters, 2007

The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal b-lactam drug. Thi... more The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal b-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-b-lactamase BcII. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind BcII, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-b-lactamases to inactive monobactams and give some clues for inhibitor design.

Research paper thumbnail of High-Level Biosynthesis of the Anteiso-C 17 Isoform of the Antibiotic Mycosubtilin in Bacillus subtilis and Characterization of Its Candidacidal Activity

Applied and Environmental Microbiology, 2009

High-level production (880 mg liter −1 ) and isolation of the anteiso-C 17 isoform of the lipopep... more High-level production (880 mg liter −1 ) and isolation of the anteiso-C 17 isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp.

Research paper thumbnail of The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Journal of Biological Chemistry, 2003

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-␤-lacta... more Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-␤-lactamases, enzymes that mediate resistance to ␤-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-␤-lactamase. The 113 Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113 Cd-edited 1 H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113 Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between H␣ of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.

Research paper thumbnail of Synthesis and biological evaluation of potential positron emission tomography (PET) ligands for brain visualization of dopamine D3 receptors

Arkivoc, 2006

Currently, the lack of a selective dopamine D 3 PET (positron emission tomography) radioligand fo... more Currently, the lack of a selective dopamine D 3 PET (positron emission tomography) radioligand for in vivo brain occupancy studies is problematic. Several requirements are necessary for a potential PET radioligand for visualization of a receptor into the brain: i) high affinity and selectivity for the target receptor; ii) suitable lipophilicity for both blood-brain barrier permeation and low nonspecific binding to proteins and lipids; iii) structural features that allow labelling with a positron emission isotope. In this study the synthesis and binding affinities for dopamine D 3 and D 2 receptors of several N-[4-(4-arylpiperazin-1-yl)butyl]arylcarboxamides are reported. These compounds were designed by the structural modification of the formerly reported D 3 receptor ligand N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-7-methoxy-2benzofurancarboxamide (1), with the aim to obtain a suitable lipophilicity and the structural features for labelling. In particular, both the 2,3-dichlorophenyl group and the 7-methoxy-2benzofurancarboxamide moiety were replaced by less lipophilic fragments. Among the studied compounds, derivatives N-[4-[4-(5-methoxy-2-benzisoxazolyl)piperazin-1-yl]butyl]-4-(4morpholinyl)benzamide (20), N-[4-[4-(5-methoxy-2-benzisoxazolyl)piperazin-1-yl]butyl]-4-(1Himidazol-1-yl)benzamide (21), and N-[4-[4-(5-methoxy-2-benzisoxazolyl)piperazin-1-yl]butyl]-5-(2-furanyl)-1H-pyrazole-3-carboxamide (22) displayed good D 3 receptor affinities (K i values 38, 22.6, and 21.3 nM, respectively) and were found to be inactive at D 2 receptor. Moreover, on the basis of their experimental log P values and their ability to cross the Caco-2 monolayer, compounds 20-22 are likely to permeate the blood-brain barrier, differently from compound 1.

Research paper thumbnail of G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets

Antimicrobial agents and chemotherapy, 2018

G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic a... more G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite , which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has...

Research paper thumbnail of Reinforced poly(hydroxyurethane) thermosets as high performance adhesives for aluminum substrates

Polymer Chemistry, 2017

High performance adhesives for bare aluminum are prepared by reinforcing poly(hydroxyurethane) (P... more High performance adhesives for bare aluminum are prepared by reinforcing poly(hydroxyurethane) (PHU) thermosets with (functional) nanofillers and poly(dimethylsiloxane).

Research paper thumbnail of Identification and characterization of EYK1, a key gene for erythritol catabolism in Yarrowia lipolytica

Applied Microbiology and Biotechnology, 2017

Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipo... more Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipolytica, in response to osmotic stress. This metabolite has application as food additive due to its sweetening properties. Although Y. lipolytica can produce erythritol at a high level from glycerol, it is also able to consume it as carbon source. This ability negatively affects erythritol productivity and represents a serious drawback for the development of an efficient erythritol production process. In this study, we have isolated by insertion mutagenesis a Y. lipolytica mutant unable to grow on erythritol. Genomic characterization of the latter highlighted that the mutant phenotype is directly related to the disruption of the YALI0F01606g gene. Several experimental evidences suggested that the identified gene, renamed EYK1, encodes an erythrulose kinase. The mutant strain showed an enhanced capacity to produce erythritol as compared to the wild-type strain. Moreover, in specific experimental conditions, it is also able to convert erythritol to erythrulose, another compound of biotechnological interest.

Research paper thumbnail of CG7054 solution structure

Research paper thumbnail of Breakdown of the stereospecificity of dd-peptidases and β-lactamases with thiolester substrates

Biochemical Journal, 1995

With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglyc... more With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an ...

Research paper thumbnail of Streptomyces K15 active-site serine dd-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions

Biochemical Journal, 1995

The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacte... more The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of carbonyl donors Z-R1-CONH-CHR2-COX-CHR3-COO- (where X is NH, S or O) and transfers the electrophilic group Z-R1-CONH-CHR2-CO to amino acceptors via an acyl-enzyme intermediate. Kinetic data suggest that the amino acceptor behaves as a simple alternative nucleophile at the level of the acyl-enzyme in the case of thiol ester and ester donors, and that it binds to the enzyme.carbonyl donor Michaelis complex and influences the rate of enzyme acylation by the carbonyl donor in the case of amide donors. Depending on the nature of the scissile bond, the enzyme has different requirements for substituents at positions R1, R2 and R3.

Research paper thumbnail of Direct n.m.r. evidence for substrate-induced conformational changes in a β -lactamase

Biochemical Journal, 1994

Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very... more Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum. The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r. experiments.

Research paper thumbnail of The mechanism of action of dd-peptidases: the role of tyrosine-159 in the Streptomyces R61 dd-peptidase

Biochemical Journal, 1993

Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or ... more Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding was also drastically decreased, except for the reactions with nitrocefin and methicillin. The consequences of the first mutation were more surprising, since a large proportion of the thiolesterase activity was retained, together with the penicillin-binding capacity. Conversely, the peptidase properties was severely affected. In both cases, a drastic decrease in the transferase activity was observed. The results are compared with those obtained by mutation of the corresponding residue in the class A beta-lactamase of Streptomyces albus G.

Research paper thumbnail of Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins

Biochemical Journal, 1991

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membra... more The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.

Research paper thumbnail of Chromogenic depsipeptide substrates for β-lactamases and penicillin-sensitive dd-peptidases

Biochemical Journal, 1990

Various ester and thioester derivatives of hippuric acid have been prepared which were substrates... more Various ester and thioester derivatives of hippuric acid have been prepared which were substrates of both beta-lactamases and DD-peptidases. The thioesters were more rapidly hydrolysed by nearly all the enzymes. Surprisingly, the enzymes acted rather efficiently on substrates which did not contain any chiral centre.

Research paper thumbnail of Complete ¹H, ¹⁵N, and ¹³C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid

Biomolecular NMR assignments, 2014

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and ... more β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96%) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.

Research paper thumbnail of High-Level Biosynthesis of the Anteiso-C₁₇ Isoform of the Antibiotic Mycosubtilin in Bacillus subtilis and Characterization of Its Candidacidal Activity

Research paper thumbnail of Thiolester substrates of dd-peptidases and ?-lactamases

Letters in Peptide Science, 1995

With peptide substrates, the penicillin-sensitive DD-peptidases exhibit a strict specificity for ... more With peptide substrates, the penicillin-sensitive DD-peptidases exhibit a strict specificity for D-AIa-D-Xaa Ctermini. Only glycine is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes also hydrolyse various ester and thiolester analogues of their natural substrates. Some of the thiolesters whose C-terminal leaving group exhibited an L stereochemistry were significantly hydrolysed by some of the studied enzymes, particularly by the Actinomadura R39 DD-peptidase. By contrast, the strict specificity for a D residue in the penultimate position was fully retained. The same esters and thiolesters also behaved as substrates for [3lactamases. In this case, thiolesters exhibiting L stereochemistry in the C-terminal position could also be hydrolysed, mainly by the class C and class D enzymes. But, more surprisingly, the class C Enterobacter cloacae P99 [Mactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes more efficiently than the D isomer.

Research paper thumbnail of Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

PLoS ONE, 2012

Background: Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase in... more Background: Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings: In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K D for different ligands. Native mass spectrometry was used as an alternative method for measuring K D values. Conclusions/Significance: Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20uC to pH 7.5/NaCl 100 mM/30uC, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

Research paper thumbnail of Structural basis for the broad-spectrum inhibition of metallo-β-lactamases by thiols

Organic & Biomolecular Chemistry, 2008

The development of broad-spectrum metallo-b-lactamase (MBL) inhibitors is challenging due to stru... more The development of broad-spectrum metallo-b-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.

Research paper thumbnail of DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase

Nucleic Acids Research, 2000

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of geno... more The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O 6alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O 6-methylguanine (O 6 meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.

Research paper thumbnail of A minimalistic approach to identify substrate binding features in B1 Metallo-β-lactamases

Bioorganic & Medicinal Chemistry Letters, 2007

The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal b-lactam drug. Thi... more The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal b-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-b-lactamase BcII. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind BcII, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-b-lactamases to inactive monobactams and give some clues for inhibitor design.

Research paper thumbnail of High-Level Biosynthesis of the Anteiso-C 17 Isoform of the Antibiotic Mycosubtilin in Bacillus subtilis and Characterization of Its Candidacidal Activity

Applied and Environmental Microbiology, 2009

High-level production (880 mg liter −1 ) and isolation of the anteiso-C 17 isoform of the lipopep... more High-level production (880 mg liter −1 ) and isolation of the anteiso-C 17 isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp.

Research paper thumbnail of The Inhibitor Thiomandelic Acid Binds to Both Metal Ions in Metallo-β-lactamase and Induces Positive Cooperativity in Metal Binding

Journal of Biological Chemistry, 2003

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-␤-lacta... more Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-␤-lactamases, enzymes that mediate resistance to ␤-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-␤-lactamase. The 113 Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113 Cd-edited 1 H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113 Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between H␣ of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.