A. Matagne | Université de Liège (original) (raw)

Papers by A. Matagne

Biochemical Journal, 1989

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between bet... more Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.

Biochemical Journal, 1998

β-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and relate... more β-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related β-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the β-lactam ring. Class A β-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new β-lactam compounds, which are meant to be ‘β-lactamase-stable’ or β-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne β-lactamase genes or by the acquisition of new genes coding for β-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A β-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic...

Journal of Chromatography B: Biomedical Sciences and Applications, 2000

A simple procedure is described which results in an optimised resolution in molecular sieve chrom... more A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples.

Journal of Biological Chemistry, 2008

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides ptero... more The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T P1 R.

Chemistry & Biology, 2001

Background: The stabilization of enzymes in the presence of substrates has been recognized for a ... more Background: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 L-lactamase, at temperatures above the melting point of the enzyme. Results: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K m values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steadystate and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. Conclusions: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri^Michaelis complex (ES) and acyl^enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.

Journal of Molecular Biology, 2000

A variety of techniques, including quenched-¯ow hydrogen exchange labelling monitored by electros... more A variety of techniques, including quenched-¯ow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-¯ow absorbance,¯uorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 C to 50 C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 C, it decreases above 40 C. In addition, the transient intermediate on the major folding pathway at 20 C, in which the a-domain is persistently structured in the absence of a stable b-domain, is thermally unfolded in a sigmoidal transition (T m % 40 C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast ($25 %) and slow ($75 %) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly ®tted to a sequential three-state model for the slow folding pathway. Together with previous ®ndings, these results indicate that the a-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the a-domain intermediate (the dominant factor at high temperatures). Destablization of such kinetically competent intermediate species is likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated.

Go, a member of heterotrimeric guanine nucleotide-binding proteins, is the most abundant form of ... more Go, a member of heterotrimeric guanine nucleotide-binding proteins, is the most abundant form of G protein in the central and peripheral nervous systems. Goα has a significant role in neuronal development and function but its signal transduction mechanism remains to be clarified.

Biochemical Journal, 1991

B-4000 Sart Tilman (Liege 1), Belgium, and tLaboratorium voor Microbiologie en microbiele Genetic... more B-4000 Sart Tilman (Liege 1), Belgium, and tLaboratorium voor Microbiologie en microbiele Genetica, Rijksuniversiteit-Gent, Ledeganckstraat 35, B-9000 Gent, Belgium Four f6-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of the Actinomadura R39 ,8-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the 'heterogeneous' preparations.

Biochemical Society Transactions, 2002

Biochemical Journal, 1991

The lysine-234 residue is highly conserved in fl-lactamases and in nearly all active-site-serine ... more The lysine-234 residue is highly conserved in fl-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A fl-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat value for benzylpenicillin was as high as 50 % of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat and kcat /Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

Biochemical Society Transactions, 1999

The Biochemical journal, 1991

Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analyse... more Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of Actinomadura R39 beta-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the 'heterogeneous' preparations.

Research in microbiology

The sensitivity of a bacterium to beta-lactam antibiotics depends upon the interplay between 3 in... more The sensitivity of a bacterium to beta-lactam antibiotics depends upon the interplay between 3 independent factors: the sensitivity of the essential penicillin-binding enzyme(s), the quantity and properties of the beta-lactamase(s) and the diffusion barrier that the outer-membrane of Gram-negative bacteria can represent. Those three factors can be modified by mutations or by the horizontal transfer of genes or portions of genes.

Antimicrobial agents and chemotherapy, 2001

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy... more Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Natural product reports, 1999

Biochemical Society transactions, 1999

Journal of molecular biology, Jan 17, 1998

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with ... more Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 degrees C and 50 degrees C. At 50 degrees C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 degrees C, although folding occurs significantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 degrees C, having persistent native-like structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 degrees C. In addition, the characteristic maximum in negative ellipticity and the minimum in fluorescence intensity observed in far UV CD and intrinsic fluorescence experiments at 20 degrees C, respectively, are not s...

The Biochemical journal, Jan 15, 1989

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown ... more By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).

Biochemical …, 1991

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serin... more The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

Biochemical Journal, 1989

Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between bet... more Ionic strength strongly influenced the turnover/inactivation ratio in the interaction between beta-halogenopenicillanates and some class A beta-lactamases. This suggested the stabilization of a highly charged intermediate by solvation. Those data could be interpreted on the basis of a reaction pathway where an episulphonium ion was transiently formed. The various mechanisms proposed for explaining the formation of the dihydrothiazine chromophore are discussed.

Biochemical Journal, 1998

β-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and relate... more β-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related β-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the β-lactam ring. Class A β-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new β-lactam compounds, which are meant to be ‘β-lactamase-stable’ or β-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne β-lactamase genes or by the acquisition of new genes coding for β-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A β-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic...

Journal of Chromatography B: Biomedical Sciences and Applications, 2000

A simple procedure is described which results in an optimised resolution in molecular sieve chrom... more A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples.

Journal of Biological Chemistry, 2008

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides ptero... more The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T P1 R.

Chemistry & Biology, 2001

Background: The stabilization of enzymes in the presence of substrates has been recognized for a ... more Background: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 L-lactamase, at temperatures above the melting point of the enzyme. Results: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K m values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steadystate and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. Conclusions: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri^Michaelis complex (ES) and acyl^enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.

Journal of Molecular Biology, 2000

A variety of techniques, including quenched-¯ow hydrogen exchange labelling monitored by electros... more A variety of techniques, including quenched-¯ow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-¯ow absorbance,¯uorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 C to 50 C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 C, it decreases above 40 C. In addition, the transient intermediate on the major folding pathway at 20 C, in which the a-domain is persistently structured in the absence of a stable b-domain, is thermally unfolded in a sigmoidal transition (T m % 40 C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast ($25 %) and slow ($75 %) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly ®tted to a sequential three-state model for the slow folding pathway. Together with previous ®ndings, these results indicate that the a-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the a-domain intermediate (the dominant factor at high temperatures). Destablization of such kinetically competent intermediate species is likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated.

Go, a member of heterotrimeric guanine nucleotide-binding proteins, is the most abundant form of ... more Go, a member of heterotrimeric guanine nucleotide-binding proteins, is the most abundant form of G protein in the central and peripheral nervous systems. Goα has a significant role in neuronal development and function but its signal transduction mechanism remains to be clarified.

Biochemical Journal, 1991

B-4000 Sart Tilman (Liege 1), Belgium, and tLaboratorium voor Microbiologie en microbiele Genetic... more B-4000 Sart Tilman (Liege 1), Belgium, and tLaboratorium voor Microbiologie en microbiele Genetica, Rijksuniversiteit-Gent, Ledeganckstraat 35, B-9000 Gent, Belgium Four f6-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of the Actinomadura R39 ,8-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the 'heterogeneous' preparations.

Biochemical Society Transactions, 2002

Biochemical Journal, 1991

The lysine-234 residue is highly conserved in fl-lactamases and in nearly all active-site-serine ... more The lysine-234 residue is highly conserved in fl-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A fl-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat value for benzylpenicillin was as high as 50 % of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat and kcat /Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

Biochemical Society Transactions, 1999

The Biochemical journal, 1991

Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analyse... more Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of Actinomadura R39 beta-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the 'heterogeneous' preparations.

Research in microbiology

The sensitivity of a bacterium to beta-lactam antibiotics depends upon the interplay between 3 in... more The sensitivity of a bacterium to beta-lactam antibiotics depends upon the interplay between 3 independent factors: the sensitivity of the essential penicillin-binding enzyme(s), the quantity and properties of the beta-lactamase(s) and the diffusion barrier that the outer-membrane of Gram-negative bacteria can represent. Those three factors can be modified by mutations or by the horizontal transfer of genes or portions of genes.

Antimicrobial agents and chemotherapy, 2001

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy... more Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Natural product reports, 1999

Biochemical Society transactions, 1999

Journal of molecular biology, Jan 17, 1998

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with ... more Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 degrees C and 50 degrees C. At 50 degrees C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 degrees C, although folding occurs significantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 degrees C, having persistent native-like structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 degrees C. In addition, the characteristic maximum in negative ellipticity and the minimum in fluorescence intensity observed in far UV CD and intrinsic fluorescence experiments at 20 degrees C, respectively, are not s...

The Biochemical journal, Jan 15, 1989

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown ... more By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).

Biochemical …, 1991

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serin... more The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.