bart devreese | Universiteit Gent (original) (raw)

Papers by bart devreese

European journal of biochemistry, Jul 1, 2001

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-b-lactamase CphA lea... more Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-b-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3 H leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.

PLOS ONE, Dec 31, 2014

Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vac... more Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, singledomain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2 RNAi and KIF9B RNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the

Journal of Bacteriology, 1997

Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3... more Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of beta-lactam antibiotics is similar to that of the full-size membrane-bound PBP, the truncated PBP is expected to adopt the native folded conformation. The truncated PBP3 functions as a thiolesterase. In aqueous media and in the presence of millimolar concentrations of a properly structured amino compound, it catalyzes the aminolysis of the thiolester until completion, suggesting that the penicillin-binding module of PBP3 is designed to catalyze transpeptidation reactions. In contrast, the truncated PBP3 is devoid of glycan polymerization activity on the E. coli lipid II intermediate, suggesting that the non-penicillin-binding module of PBP3 is not a transglycosylase.

Carbohydrate research, Jan 22, 2017

The carbohydrate structures of molluscan hemocyanins have recently received particular interest d... more The carbohydrate structures of molluscan hemocyanins have recently received particular interest due to their specific monosaccharide composition, as well as their immunostimulatory properties and application in clinical studies. For the first time, we investigated N-glycans of the structural subunit β-HlH of hemocyanin isolated from Helix lucorum. In total, 32 different glycans were enzymatically liberated and characterized by tandem mass spectrometry using a Q-Trap mass spectrometer. Our study revealed a highly heterogeneous mixture of glycans with composition Hex3-7HexNAc2-5MeHex0-4Pent0-1Fuc0-1. The oligosaccharide chains are mostly modified at the inner core by β1-2-linked xylose to β-mannose, by α1-6-fucosylation of the innermost GlcNAc residue (the Asn-bound GlcNAc), and by methylation. The glycans of β-HlH mainly contain a terminal MeHex residue; in some cases even two, three or four of these residues occur. Several carbohydrate chains in β-HlH are core-fucosylated without Xy...

PROTEOMICS, 2005

Shewanella oneidensis MR-1 is a Gram-negative, facultative aerobic bacterium, able to respire a v... more Shewanella oneidensis MR-1 is a Gram-negative, facultative aerobic bacterium, able to respire a variety of electron acceptors. Due to its capability to reduce solid ferric iron, S. oneidensis plays an important role in microbially induced corrosion of metal surfaces. Since this requires cellular adhesion to the metal surface, biofilm growth is an essential feature of this process. The goal of this work was to compare the global protein expression patterns of sessile and planktonic grown S. oneidensis cells by two-dimensional (2-D) gel electrophoresis. Mass spectrometry was used as an identification tool of the differentially expressed proteins. An IPG strip of pH 3-10 as well as pH 4-7 was applied for iso-electrofocusing. Analysis of the 2-D patterns pointed out a total of 59 relevant spots. Among these proteins, we highlight the involvement of a protein annotated as an agglutination protein (AggA). AggA is a TolC-like protein which is presumably part of an ABC transporter. Another differentially expressed protein is RibB, an enzyme of the riboflavin biosynthesis pathway. Riboflavin is the precursor molecule of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and may be necessary for the altered respiratory properties of the biofilm cells versus planktonic cells. Some proteins that were identified indicate an anaerobic state of the biofilm. This anaerobic way of living affects the energy gaining pathways of the cell and is reflected by the presence of several proteins, including those of a heme-utilization system.

Protein Science, 1993

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothi... more The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospiru halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Nutl. Acud. Sci. USA 86,6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 11 1 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (k0.5 Da); the cysteine residue to which it is bound is at sequence position 69.

Molecular Microbiology, 2000

Different strains of Bacillus were screened for their ability to hydrolyse d‐alanyl‐p‐nitroanilid... more 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, e...

Molecular and Cellular Endocrinology, 1998

Monoclonal antibodies (Mabs) specifically recognizing the chicken pituitary corticotropes were us... more Monoclonal antibodies (Mabs) specifically recognizing the chicken pituitary corticotropes were used to isolate a population of closely related peptides from crude chicken pituitary extracts. A homogeneous N-terminal sequence homologous to the extreme N-terminus of mammalian and amphibian pro-opiomelanocortin (POMC) was revealed. Further physicochemical analysis proved the existence of a series of C-terminally truncated peptides including 3 major molecular species corresponding to Ser1-Gly64, Ser1-Arg73 and Ser1-Gly105 respectively. The two latter molecules were shown to be N-glycosylated at position Asn67, with mass spectrometric data indicating a carbohydrate structure of the oligomannose 5 type, in addition to two more complex structures. No evidence was found in favour of O-glycosylation on Ser47. Degenerated PCR primers were deduced from the above protein sequence and from the known chicken adrenocorticotropic hormone (ACTH) sequence. The nucleotide sequence obtained by reversed transcription PCR (RT-PCR) completely confirmed the new amino acid sequence data including pro-gamma-MSH, the joining peptide and ACTH.

Journal of the American Society for Mass Spectrometry, 2006

Metallo-␤-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate ␤-lact... more Metallo-␤-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate ␤-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-␤-lactamase because it degrades only carbapenems efficiently and is only active when it has one zinc ion bound. A zinc titration experiment was used to study the zinc affinity of the wild-type and of several mutant CphA enzymes. It shows that a second Zn 2ϩ is also bound at high ion concentrations. All samples were analyzed using mass spectrometry in combination with an automated nanoESI source. The metal-free enzyme has a bimodal charge distribution indicative of two conformational states. A completely folded enzyme is detected when the apo-enzyme has bound the first zinc. Intensity ratios of the different enzyme forms were used to deduce the zinc affinities. CphA enzymes mutated in metal ligands show decreased zinc affinity compared to wild-type, especially D120 mutants.

Journal of Endocrinology, 2001

Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal clea... more Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. T...

JBIC Journal of Biological Inorganic Chemistry, 2002

The present study describes the cloning, isolation, and thorough biochemical characterization of ... more The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacillus pasteurii, a novel protein putatively involved in the transport of Ni in the urease assembly process. A DNA fragment of the B. pasteurii urease operon, containing all four accessory genes (ureE, ureF, ureG, and ureD) required for the incorporation of Ni ions into the active site of urease, was cloned, sequenced, and analyzed. B. pasteurii ureE was cloned, and the UreE protein (BpUreE) was over-expressed and purified to homogeneity. The identity of the recombinant protein was determined by N-and C-terminal sequencing and by mass spectrometry. BpUreE has a chain length of 147 amino acids, and features a pI value of 4.7. As isolated, BpUreE contains one Zn(II) ion per dimer, while no Ni(II) is present, as shown by mass spectrometry and atomic absorption spectroscopy. BpUreE behaves as a dimer independently of the presence of Zn(II), as shown by gel filtration and mass spectrometry. Paramagnetic NMR spectroscopy on concentrated (2 mM) UreE solutions reveals a one Ni atom per tetramer stoichiometry, with the Ni(II) ion bound to histidines in an octahedral coordination environment. BpUreE has a high sequence similarity with UreE proteins isolated from different biological sources, while no sequence homology is observed with proteins belonging to different classes. In particular, BpUreE is most similar to UreE from Bacillus halodurans (55% identity). A multiple sequence alignment reveals the presence of four strictly conserved residues (Leu55, Gly97, Asn98, His100; BpUreE numbering), in addition to position 115, conservatively occupied by an Asp or a Glu residue. Several secondary structure elements, including a babbab ''ferredoxin-like'' motif, are highly conserved throughout the UreE sequences. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.

Journal of Biological Chemistry, 2004

Journal of Biological Chemistry, 2001

Journal of Biological Chemistry, 2005

Among class B β-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity ... more Among class B β-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion. Abreviations: WT, wild type; CD, circular dichroism In vivo, class B β-lactamases (1) require one or two zinc ions as enzymatic cofactors. By efficiently catalyzing the hydrolysis of the β-lactam amide bond, these enzymes play a key role in bacterial resistance to this group of antibiotics. The metallo-β-lactamase family has been divided into three different subclasses, B1, B2, and B3, on the basis of sequence similarities (2, 3). The CphA metallo-β-lactamase produced by Aeromonas hydrophila belongs to subclass B2. It is characterized by a uniquely narrow specificity profile. CphA efficiently hydrolyzes only carbapenems and shows very poor activity against penicillins and cephalosporins, a behavior in contrast to that of metallo-β-lactamases of subclasses B1and B3, which usually exhibit very broad activity spectra against nearly all β-lactam compounds, with the exception of monobactams (4, 5). Moreover, in contrast to the BcII (Bacillus cereus) and CcrA (Bacteroides fragilis) enzymes belonging to the B1 subclass, and in general to most other metallo-β-lactamases, CphA exhibits a maximum activity as a mono-zinc enzyme. The presence of a Zn 2+ ion in a second low affinity binding site non-competitively inhibits the enzyme with a K i value of 46 µM at pH 6.5 (6). Recently, the structure of the mono-zinc CphA enzyme has been solved by x-ray crystallography (7). Similar to the known structures of metallo-β-lactamases of subclasses B1 (BcII (8), CcrA (9), IMP-I (10), BlaB (11)) and B3 (L1 (12) and FEZ-1 (13)), the x-ray structure of CphA highlights an αββα sandwich with two central β-sheets and α-helices on the external faces. The active site is located at the bottom of the β-sheet core. In agreement with previous spectroscopic results (14, 15) and site-directed mutagenesis studies (16), these structural data show that the sole Zn 2+ ion resides in the Asp 120-Cys 221-His 263 site of the A. hydrophila metallo-βlactamase. In the di-zinc form of subclass B1, the zinc ions occupy both the His 116 , His 118 , and His 196 and the Asp 120 , Cys 221 , and His 263 sites (see Fig. 1). The histidine residue in position 116 in most metallo-β-lactamases is replaced by an asparagine residue in CphA (2, 17). This Asn-116 residue is not responsible for the narrow substrate profile of CphA, because the activity of the N116H mutant (where the three-histidine site found in most metallo-β-lactamases is recreated) against nitrocefin, benzyl-penicillin, and cephaloridine, although increased, remains rather low (16). Moreover, the K D 2 values are similar for the N116S mutant and the wild-type enzyme, indicating that Asn 116 does not participate in the binding of the second metal ion.

Journal of Biological Chemistry, 2008

Gene, 2011

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many mol... more Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α D-HlH, α N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.

Extremophiles, 2000

A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that ex... more A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 Ϯ 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0°-20°C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

Chemistry & Biology, 2003

sequences. Subclass B1 is the largest and contains three well-studied ␤-lactamases: BcII from Bac... more sequences. Subclass B1 is the largest and contains three well-studied ␤-lactamases: BcII from Bacillus cereus [2-4], CcrA from Bacteroides fragilis [5-8], and IMP-1 from Pseudomonas aeruginosa [9-11]. The subclass B2 prototypical enzyme is CphA from Aeromonas

Cellular and Molecular Life Sciences (CMLS), 2003

The CphA metallo-β-actamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Max... more The CphA metallo-β-actamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asnl 16 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.