Tatyana Shcherbakova - Academia.edu (original) (raw)
Papers by Tatyana Shcherbakova
Acta naturae, Aug 3, 2023
КРАТКИЕ СООБЩЕНИЯ РЕФЕРАТ В результате компьютерного скрининга библиотеки сульфозамещенных соедин... more КРАТКИЕ СООБЩЕНИЯ РЕФЕРАТ В результате компьютерного скрининга библиотеки сульфозамещенных соединений выявлены молекулы, способные связываться в активном центре транскетолазы из Mycobacterium tuberculosis. Осуществлена экспериментальная проверка ингибиторной активности наиболее перспективного соединения STK045765 в отношении высокоочищенного препарата рекомбинантного фермента. Показано, что молекула STK045765 конкурирует за участок связывания пирофосфатной группы кофактора тиаминдифосфата и в микромолярных концентрациях способна подавлять активность микобактериальной транскетолазы. Обнаруженный фурансульфонатный скаффолд может служить основой для создания противотуберкулезных препаратов. КЛЮЧЕВЫЕ СЛОВА сульфонаты, сульфогруппа, ингибиторы, тиаминдифосфат, транскетолаза, микобактерии. СПИСОК СОКРАЩЕНИЙ mbТК-транскетолаза микобактерий.
FEBS Journal, Sep 9, 2008
The 2-oxoglutarate dehydrogenase complex (OGDHC) is a key regulator of a branch point in the tric... more The 2-oxoglutarate dehydrogenase complex (OGDHC) is a key regulator of a branch point in the tricarboxylic acid cycle. It belongs to the family of 2-oxo acid dehydrogenase complexes which comprise multiple copies of the three catalytic enzyme components: E1, thiamine diphosphate (ThDP)-dependent 2-oxo acid dehydrogenase (in OGDHC it is E1o); E2, dihydrolipoyl acyltransferase with the covalently bound lipoic acid Keywords 2-oxoglutarate dehydrogenase isoenzyme; mitochondrial membrane; multienzyme complex; thiamine; tricarboxylic acid cycle
<p>Experimental characterization of the wild type (WT) <i>Ec</i>PA and its Dβ48... more <p>Experimental characterization of the wild type (WT) <i>Ec</i>PA and its Dβ484N mutant.</p
<p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three i... more <p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three independent MD runs. The mean and standard deviation are shown in angstroms. N<sub>res</sub> – number of amino acid residues in a structural domain. Δ<sub>RMSD</sub> = RMSD<sub>pH10</sub> – RMSD<sub>pH7.5</sub> and <i>f</i> = <i>f</i>(RMSD<sub>pH7.5</sub> ≥ RMSD<sub>pH10</sub>) were calculated as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100643#s4" target="_blank">Methods</a>, section 2. Lower <i>f</i> values indicate a significant destabilizing effect of the alkaline pH compared to neutral conditions while Δ<sub>RMSD</sub> estimates the degree of destabilization.</p
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2008
Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-se... more Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.
Applied and Environmental Microbiology, 2006
The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In t... more The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to β-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the ...
Frontiers in Pharmacology
7-Methylguanine (7-MG) competitively inhibits the DNA repair enzyme poly(ADP-ribose) polymerase (... more 7-Methylguanine (7-MG) competitively inhibits the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and RNA-modifying enzyme tRNA-guanine transglycosylase (TGT) and represents a potential anticancer drug candidate. Furthermore, as a natural compound, it could escape the serious side effects characteristic for approved synthetic PARP inhibitors. Here we present a comprehensive study of toxicological and carcinogenic properties of 7-MG. It was demonstrated that 7-MG does not induce mutations or structural chromosomal abnormalities, and has no blastomogenic activity. A treatment regimen with 7-MG has been established in mice (50 mg/kg per os, 3 times per week), exerting no adverse effects or changes in morphology. Preliminary data on the 7-MG anticancer activity obtained on transplantable tumor models support our conclusions that 7-MG can become a promising new component of chemotherapy.
<p>Positions are ranked in a declined statistical significance (see <a href="http:/... more <p>Positions are ranked in a declined statistical significance (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100643#pone.0100643-Suplatov1" target="_blank">[68]</a> for details). PA – penicillin acylases, GAs – glutaryl-7-aminocephalosporanic acid acylases, AHLs (PvdQ) – N-acyl homoserine lactone acylases PvdQ. The most frequently occurring amino acids are shown for every subfamily.</p
<p>Results are shown for <i>Ec</i>PA and its Dβ484N mutant at different pH. Eac... more <p>Results are shown for <i>Ec</i>PA and its Dβ484N mutant at different pH. Each curve is averaged over three independent MD trajectories.</p
<p>(A) Glutarylamidase from <i>Pseudomonas sp</i>., (B) N-acyl homoserine lacto... more <p>(A) Glutarylamidase from <i>Pseudomonas sp</i>., (B) N-acyl homoserine lactone acylase PvdQ from <i>Pseudomonas aeruginosa</i>, and (C) penicillin acylase from <i>Alcaligenes faecalis</i>.</p
Moscow University Chemistry Bulletin, 2008
The gene of penicillin acylase (PA) from Escherichia coli has been cloned from a PA producer stra... more The gene of penicillin acylase (PA) from Escherichia coli has been cloned from a PA producer strain that is an analogue of strain ATCC 11105. Optimization of the cultivation conditions made it possible to obtain up to 130 mg of active enzyme per liter of culture broth. A number of single, double, and triple mutants were obtained by the method of site-specific mutagenesis using PCR. As a result of isolation and purification procedures, homogeneous preparations of the wild-type enzyme and its mutants were obtained. Studies showed that (1) the obtained enzymes have the correctly folded structure; (2) complexing agents and metal cations do not inhibit their catalytic activity; (3) mutant PAs, like the wild type, are efficiently inactivated by phenylmethylsulfonyl fluoride (PMSF), which makes it possible to titrate their active sites; and (4) the obtained mutants are characterized by a greater specificity constant in the reaction of hydrolysis of a colorimetric substrate; however, they are inferior to the wild type in the synthesis of ampicillin by acyl transfer.
<p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three i... more <p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three independent MD runs. The mean and standard deviation are shown in angstroms. N<sub>res</sub> – number of amino acid residues in a structural unit. Δ<sub>RMSD</sub> = RMSD<sub>Dβ484N</sub> – RMSD<sub>WT</sub> and <i>f</i> = <i>f</i>(RMSD<sub>Dβ484N</sub> ≥ RMSD<sub>WT</sub>) were calculated as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100643#s4" target="_blank">Methods</a>, section 2. Lower <i>f</i> values indicate a significant stabilizing effect of the mutation compared to the wild type enzyme while Δ<sub>RMSD</sub> estimates the degree of stabilization.</p
Journal of Molecular Catalysis B: Enzymatic, 2004
Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis an... more Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis and aminolysis of d-phenylglycine amide. Inactivation was very sensitive to the d-phenylglycine amide concentration: at pH 9.5, 25°C and 400mM substrate, penicillin acylase lost more than 90% of its initial catalytic activity in half an hour, in the presence of 100mM substrate, 50% of the initial activity in two hours, whereas in the absence of substrate, no significant enzyme inactivation was observed in three hours. Observed enzyme inactivation limits use of high acyl donor concentrations at penicillin acylase-catalyzed peptide synthesis.
Journal of Molecular Catalysis B: Enzymatic, 2015
PLoS ONE, 2014
Protein stability provides advantageous development of novel properties and can be crucial in aff... more Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluβ482 and Aspβ484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspβ484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dβ484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2002
Nucleophile reactivity of two most known nuclei of penicillins and cephalosporins, 6-aminopenicil... more Nucleophile reactivity of two most known nuclei of penicillins and cephalosporins, 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids, was quantitatively characterized. In penicillin acylase (PA)-catalyzed acyl transfer reactions the relative reactivity of the added nucleophile compared to the water (i.e. nucleophile reactivity) is defined by two complex kinetic parameters beta(0) and gamma, and depends on the nucleophile concentration. In turn, parameters beta(0) and gamma were shown to be dependent on the structure of both reactants involved: nucleophile and acyl donor. Analysis of the kinetic scheme revealed that nucleophile reactivity is one of a few key parameters controlling efficiency of PA-catalyzed acyl transfer to the added nucleophile in an aqueous medium. Computation of the maximum nucleophile conversion to the product using determined nucleophile reactivity parameters in the synthesis of three different antibiotics, ampicillin, amoxicillin and cephalexin, showed good correlation with the results of corresponding synthetic experiments. Suggested approach can be extended to the quantitative description and optimization of PA-catalyzed acyl transfer reactions in a wide range of experimental conditions.
Journal of Molecular Catalysis B: Enzymatic, 2004
Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis an... more Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis and aminolysis of d-phenylglycine amide. Inactivation was very sensitive to the d-phenylglycine amide concentration: at pH 9.5, 25°C and 400mM substrate, penicillin acylase lost more than 90% of its initial catalytic activity in half an hour, in the presence of 100mM substrate, 50% of the initial activity in two hours, whereas in the absence of substrate, no significant enzyme inactivation was observed in three hours. Observed enzyme inactivation limits use of high acyl donor concentrations at penicillin acylase-catalyzed peptide synthesis.
Acta naturae, Aug 3, 2023
КРАТКИЕ СООБЩЕНИЯ РЕФЕРАТ В результате компьютерного скрининга библиотеки сульфозамещенных соедин... more КРАТКИЕ СООБЩЕНИЯ РЕФЕРАТ В результате компьютерного скрининга библиотеки сульфозамещенных соединений выявлены молекулы, способные связываться в активном центре транскетолазы из Mycobacterium tuberculosis. Осуществлена экспериментальная проверка ингибиторной активности наиболее перспективного соединения STK045765 в отношении высокоочищенного препарата рекомбинантного фермента. Показано, что молекула STK045765 конкурирует за участок связывания пирофосфатной группы кофактора тиаминдифосфата и в микромолярных концентрациях способна подавлять активность микобактериальной транскетолазы. Обнаруженный фурансульфонатный скаффолд может служить основой для создания противотуберкулезных препаратов. КЛЮЧЕВЫЕ СЛОВА сульфонаты, сульфогруппа, ингибиторы, тиаминдифосфат, транскетолаза, микобактерии. СПИСОК СОКРАЩЕНИЙ mbТК-транскетолаза микобактерий.
FEBS Journal, Sep 9, 2008
The 2-oxoglutarate dehydrogenase complex (OGDHC) is a key regulator of a branch point in the tric... more The 2-oxoglutarate dehydrogenase complex (OGDHC) is a key regulator of a branch point in the tricarboxylic acid cycle. It belongs to the family of 2-oxo acid dehydrogenase complexes which comprise multiple copies of the three catalytic enzyme components: E1, thiamine diphosphate (ThDP)-dependent 2-oxo acid dehydrogenase (in OGDHC it is E1o); E2, dihydrolipoyl acyltransferase with the covalently bound lipoic acid Keywords 2-oxoglutarate dehydrogenase isoenzyme; mitochondrial membrane; multienzyme complex; thiamine; tricarboxylic acid cycle
<p>Experimental characterization of the wild type (WT) <i>Ec</i>PA and its Dβ48... more <p>Experimental characterization of the wild type (WT) <i>Ec</i>PA and its Dβ484N mutant.</p
<p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three i... more <p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three independent MD runs. The mean and standard deviation are shown in angstroms. N<sub>res</sub> – number of amino acid residues in a structural domain. Δ<sub>RMSD</sub> = RMSD<sub>pH10</sub> – RMSD<sub>pH7.5</sub> and <i>f</i> = <i>f</i>(RMSD<sub>pH7.5</sub> ≥ RMSD<sub>pH10</sub>) were calculated as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100643#s4" target="_blank">Methods</a>, section 2. Lower <i>f</i> values indicate a significant destabilizing effect of the alkaline pH compared to neutral conditions while Δ<sub>RMSD</sub> estimates the degree of destabilization.</p
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2008
Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-se... more Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.
Applied and Environmental Microbiology, 2006
The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In t... more The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to β-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the ...
Frontiers in Pharmacology
7-Methylguanine (7-MG) competitively inhibits the DNA repair enzyme poly(ADP-ribose) polymerase (... more 7-Methylguanine (7-MG) competitively inhibits the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) and RNA-modifying enzyme tRNA-guanine transglycosylase (TGT) and represents a potential anticancer drug candidate. Furthermore, as a natural compound, it could escape the serious side effects characteristic for approved synthetic PARP inhibitors. Here we present a comprehensive study of toxicological and carcinogenic properties of 7-MG. It was demonstrated that 7-MG does not induce mutations or structural chromosomal abnormalities, and has no blastomogenic activity. A treatment regimen with 7-MG has been established in mice (50 mg/kg per os, 3 times per week), exerting no adverse effects or changes in morphology. Preliminary data on the 7-MG anticancer activity obtained on transplantable tumor models support our conclusions that 7-MG can become a promising new component of chemotherapy.
<p>Positions are ranked in a declined statistical significance (see <a href="http:/... more <p>Positions are ranked in a declined statistical significance (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100643#pone.0100643-Suplatov1" target="_blank">[68]</a> for details). PA – penicillin acylases, GAs – glutaryl-7-aminocephalosporanic acid acylases, AHLs (PvdQ) – N-acyl homoserine lactone acylases PvdQ. The most frequently occurring amino acids are shown for every subfamily.</p
<p>Results are shown for <i>Ec</i>PA and its Dβ484N mutant at different pH. Eac... more <p>Results are shown for <i>Ec</i>PA and its Dβ484N mutant at different pH. Each curve is averaged over three independent MD trajectories.</p
<p>(A) Glutarylamidase from <i>Pseudomonas sp</i>., (B) N-acyl homoserine lacto... more <p>(A) Glutarylamidase from <i>Pseudomonas sp</i>., (B) N-acyl homoserine lactone acylase PvdQ from <i>Pseudomonas aeruginosa</i>, and (C) penicillin acylase from <i>Alcaligenes faecalis</i>.</p
Moscow University Chemistry Bulletin, 2008
The gene of penicillin acylase (PA) from Escherichia coli has been cloned from a PA producer stra... more The gene of penicillin acylase (PA) from Escherichia coli has been cloned from a PA producer strain that is an analogue of strain ATCC 11105. Optimization of the cultivation conditions made it possible to obtain up to 130 mg of active enzyme per liter of culture broth. A number of single, double, and triple mutants were obtained by the method of site-specific mutagenesis using PCR. As a result of isolation and purification procedures, homogeneous preparations of the wild-type enzyme and its mutants were obtained. Studies showed that (1) the obtained enzymes have the correctly folded structure; (2) complexing agents and metal cations do not inhibit their catalytic activity; (3) mutant PAs, like the wild type, are efficiently inactivated by phenylmethylsulfonyl fluoride (PMSF), which makes it possible to titrate their active sites; and (4) the obtained mutants are characterized by a greater specificity constant in the reaction of hydrolysis of a colorimetric substrate; however, they are inferior to the wild type in the synthesis of ampicillin by acyl transfer.
<p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three i... more <p>Root mean square deviation (RMSD) for each enzyme variant has been averaged over three independent MD runs. The mean and standard deviation are shown in angstroms. N<sub>res</sub> – number of amino acid residues in a structural unit. Δ<sub>RMSD</sub> = RMSD<sub>Dβ484N</sub> – RMSD<sub>WT</sub> and <i>f</i> = <i>f</i>(RMSD<sub>Dβ484N</sub> ≥ RMSD<sub>WT</sub>) were calculated as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100643#s4" target="_blank">Methods</a>, section 2. Lower <i>f</i> values indicate a significant stabilizing effect of the mutation compared to the wild type enzyme while Δ<sub>RMSD</sub> estimates the degree of stabilization.</p
Journal of Molecular Catalysis B: Enzymatic, 2004
Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis an... more Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis and aminolysis of d-phenylglycine amide. Inactivation was very sensitive to the d-phenylglycine amide concentration: at pH 9.5, 25°C and 400mM substrate, penicillin acylase lost more than 90% of its initial catalytic activity in half an hour, in the presence of 100mM substrate, 50% of the initial activity in two hours, whereas in the absence of substrate, no significant enzyme inactivation was observed in three hours. Observed enzyme inactivation limits use of high acyl donor concentrations at penicillin acylase-catalyzed peptide synthesis.
Journal of Molecular Catalysis B: Enzymatic, 2015
PLoS ONE, 2014
Protein stability provides advantageous development of novel properties and can be crucial in aff... more Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluβ482 and Aspβ484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspβ484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dβ484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2002
Nucleophile reactivity of two most known nuclei of penicillins and cephalosporins, 6-aminopenicil... more Nucleophile reactivity of two most known nuclei of penicillins and cephalosporins, 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids, was quantitatively characterized. In penicillin acylase (PA)-catalyzed acyl transfer reactions the relative reactivity of the added nucleophile compared to the water (i.e. nucleophile reactivity) is defined by two complex kinetic parameters beta(0) and gamma, and depends on the nucleophile concentration. In turn, parameters beta(0) and gamma were shown to be dependent on the structure of both reactants involved: nucleophile and acyl donor. Analysis of the kinetic scheme revealed that nucleophile reactivity is one of a few key parameters controlling efficiency of PA-catalyzed acyl transfer to the added nucleophile in an aqueous medium. Computation of the maximum nucleophile conversion to the product using determined nucleophile reactivity parameters in the synthesis of three different antibiotics, ampicillin, amoxicillin and cephalexin, showed good correlation with the results of corresponding synthetic experiments. Suggested approach can be extended to the quantitative description and optimization of PA-catalyzed acyl transfer reactions in a wide range of experimental conditions.
Journal of Molecular Catalysis B: Enzymatic, 2004
Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis an... more Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis and aminolysis of d-phenylglycine amide. Inactivation was very sensitive to the d-phenylglycine amide concentration: at pH 9.5, 25°C and 400mM substrate, penicillin acylase lost more than 90% of its initial catalytic activity in half an hour, in the presence of 100mM substrate, 50% of the initial activity in two hours, whereas in the absence of substrate, no significant enzyme inactivation was observed in three hours. Observed enzyme inactivation limits use of high acyl donor concentrations at penicillin acylase-catalyzed peptide synthesis.