RESUMO
The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared with that of the wild-type. Crystallographic experiments, designed to characterize these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.
Assuntos
Acinetobacter calcoaceticus , Proteínas de Bactérias , Glucose 1-Desidrogenase , Peróxido de Hidrogênio , Acinetobacter calcoaceticus/enzimologia , Acinetobacter calcoaceticus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Glucose/metabolismo , Glucose 1-Desidrogenase/genética , Glucose 1-Desidrogenase/metabolismo , Peróxido de Hidrogênio/metabolismo , Mutação , Cofator PQQ/metabolismo , Especificidade por SubstratoRESUMO
The four mammalian peroxidases (myeloperoxidase, eosinophilperoxidase, lactoperoxidase, and thyroid peroxidase) are widely studied in the literature. They catalyze the formation of antimicrobial compounds and participate in innate immunity. Owing to their properties, they are used in many biomedical, biotechnological, and agro-food applications. We decided to look for an enzyme that is easiest to produce and much more stable at 37 °C than mammalian peroxidases. To address this question, a peroxidase from Rhodopirellula baltica, identified by bioinformatics tools, was fully characterized in this study. In particular, a production and purification protocol including the study of heme reconstitution was developed. Several activity tests were also performed to validate the hypothesis that this peroxidase is a new homolog of mammalian myeloperoxidase. It has the same substrate specificities as the human one and accepts I-, SCN-, Br-, and Cl- as (pseudo-) halides. It also exhibits other auxiliary activities such as catalase and classical peroxidase activities, and it is very stable at 37 °C. Finally, this bacterial myeloperoxidase can kill the Escherichia coli strain ATCC25922, which is usually used to perform antibiograms.
RESUMO
The ability to switch on the activity of an enzyme through its spontaneous reconstitution has proven to be a valuable tool in fundamental studies of enzyme structure/reactivity relationships or in the design of artificial signal transduction systems in bioelectronics, synthetic biology, or bioanalytical applications. In particular, those based on the spontaneous reconstitution/activation of the apo-PQQ-dependent soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed. However, the reconstitution mechanism of sGDH with its two cofactors, i.e., pyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here is to elucidate this mechanism by stopped-flow kinetics under single-turnover conditions. The reconstitution of sGDH exhibited biphasic kinetics, characteristic of a square reaction scheme associated with two activation pathways. From a complete kinetic analysis, we were able to fully predict the reconstitution dynamics and also to demonstrate that when PQQ first binds to apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed position at the bottom of the enzyme binding site, thereby greatly slowing down the reconstitution rate of sGDH. This slow calcium insertion may purposely be accelerated by providing more flexibility to the Ca2+ binding loop through the specific mutation of the calcium-coordinating P248 proline residue, reducing thus the kinetic barrier to calcium ion insertion. The dynamic nature of the reconstitution process is also supported by the observation of a clear loop shift and a reorganization of the hydrogen-bonding network and van der Waals interactions observed in both active sites of the apo and holo forms, a structural change modulation that was revealed from the refined X-ray structure of apo-sGDH (PDB: 5MIN).
RESUMO
Here, we describe how poor exam results of undergraduate students enrolled in an enzymology course at the University of Bordeaux were improved through the introduction of 'clickers' as an audience response system. By using clickers only in a small-group tutorial element of a large theoretical course, we observed an improvement in exam scores that resulted in a lower failure rate for the course. Furthermore, students of all abilities were found to benefit from their use. Students reported better retention of both lecture and tutorial content. An analysis of how clickers were employed within the tutorials indicated that the use of clickers to promote discussion and impart knowledge likely resulted in a moderate improvement of exam scores. We hypothesize that students were more prepared for exams through greater reflection of exam questions, resulting in an enhanced ability to retrieve memorized information and apply it within a time-limited exam setting.
Assuntos
Desempenho Acadêmico/estatística & dados numéricos , Bioquímica/educação , Ensino , FrançaRESUMO
We present a new heterogeneous biocatalyst based on the grafting of Bilirubin Oxidase from Bacillus pumilus into macrocellular Si(HIPE) materials dedicated to water treatment. Due to the host intrinsic high porosity and monolithic character, on-line catalytic process is reached. We thus used this biocatalyst toward uni-axial flux decolorizations of Congo Red and Remazol Brilliant Blue (RBBR) at two different pH (4 and 8.2), both in presence or absence of redox mediator. In absence of redox mediators, 40% decolorization efficiency was reached within 24 h at pH 4 for Congo Red and 80% for RBBR at pH 8.2 in 24 h. In presence of 10µM ABTS, it respectively attained 100% efficiency after 2h and 12h. We have also demonstrated that non-toxic species were generated upon dyes decolorization. These results show that unlike laccases, this new biocatalyst exhibits excellent decolorization properties over a wide range of pH. Beyond, this enzymatic-based heterogeneous catalyst can be reused during two months being simply stored at room temperature while maintaining its decolorization efficiency. This study shows that this biocatalyst is a promising and robust candidate toward wastewater treatments, both in acidic and alkaline conditions where in the latter efficient decolorization strategies were still missing.
Assuntos
Bacillus pumilus/enzimologia , Biodegradação Ambiental , Corantes/química , Corantes/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Dióxido de Silício/química , Catálise , Sistemas On-Line , OxirreduçãoRESUMO
Small hydrophobic membrane proteins or proteins with hydrophobic domains are often difficult to produce in bacteria. The cell-free expression system was found to be a very good alternative for the expression of small hydrophobic subunits of the yeast ATP-synthase, such as subunits e, g, k, i, f and the membrane domain of subunit 4, proteins that are suspected to play a role in the stability of ATP-synthase dimers. All of these proteins could be produced in milligrams amounts using the cell-free "precipitate mode" and were successfully solubilized in the presence of lysolipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol. Purified proteins were also found suitable for structural investigations. An example is given with the NMR backbone assignment of the isotopically labeled subunit g. Protocols are also described for raising specific polyclonal antibodies against overexpressed cell-free proteins.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Sistema Livre de Células , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , ATPases Mitocondriais Próton-Translocadoras/química , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Subunidades Proteicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Multicopper oxidases (MCOs) catalyzed two half reactions (linked by an intramolecular electron transfer) through a Ping-Pong mechanism: the substrate oxidation followed by the O2 reduction. MCOs have been characterized in details in solution or immobilized on electrode surfaces. The nature of the rate-limiting steps, which is controversial in the literature, is discussed in this mini review for both cases. Deciphering such rate-limiting steps is of particular importance to efficiently use MCOs in any applications requiring the reduction of O2 to water.
RESUMO
Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase (BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%.
Assuntos
Bacillus pumilus/genética , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas Recombinantes/metabolismo , Bacillus pumilus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Enzymatic biofuel cells (BFCs) may power implanted medical devices and will rely on the use of glucose and O2 available in human bodily fluids. Other than well-established experiments in aqueous buffer, little work has been performed in whole human blood because it contains numerous inhibiting molecules. Here, we tested our BFCs in 30 anonymized, random and disease-free whole human blood samples. We show that by designing our anodic and cathodic bioelectrocatalysts with osmium based redox polymers and home-made enzymes we could reach a high selectivity and biofunctionnality. After optimization, BFCs generate power densities directly proportional to the glycaemia of human blood and reached a maximum power density of 129µWcm(-2) at 0.38V vs. Ag/AgCl at 8.22mM glucose. This is to our knowledge the highest power density attained so far in human blood and open the way for the powering of integrated medical feedback loops.
Assuntos
Fontes de Energia Bioelétrica , Glicemia/metabolismo , Eletricidade , Oxigênio/metabolismo , Fontes de Energia Bioelétrica/microbiologia , Eletrodos , Desenho de Equipamento , Glucose 1-Desidrogenase/metabolismo , Humanos , Magnaporthe/enzimologia , Osmio/química , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Oxigênio/sangue , Polímeros/química , Pseudomonadaceae/enzimologiaRESUMO
In the 5-8 mM glucose concentration range, of particular interest for diabetes management, glucose oxidase bioelectrodes are O2 dependent, which decrease their efficiencies. By replacing the natural cofactor of glucose oxidase, we succeeded in turning an O2 sensitive bioelectrode into an almost insensitive one.
Assuntos
Eletrodos , Glucose Oxidase/química , Glucose/análise , Oxigênio/química , Aspergillus niger/enzimologia , Calibragem , CinéticaRESUMO
A highly sensitive electroanalytical method for determination of PQQ in solution down to subpicomolar concentrations is proposed. It is based on the heterogeneous reconstitution of the PQQ-dependent glucose dehydrogenase (PQQ-GDH) through the specific binding of its pyrroloquinoline quinone (PQQ) cofactor to the apoenzyme anchored on an electrode surface. It is shown from kinetics analysis of both the enzyme catalytic responses and enzyme surface-reconstitution process (achieved by cyclic voltammetry under redox-mediated catalysis) that the selected immobilization strategy (i.e., through an avidin/biotin linkage) is well-suited to immobilize a nearly saturated apoenzyme monolayer on the electrode surface with an almost fully preserved PQQ binding properties and catalytic activity. From measurement of the overall rate constants controlling the steady-state catalytic current responses of the surface-reconstituted PQQ-GDH and determination of the PQQ equilibrium binding (Kb = 2.4 × 10(10) M(-1)) and association rate (kon = 2 × 10(6) M(-1) s(-1)) constants with the immobilized apoenzyme, the analytical performances of the method could be rationally evaluated, and the signal amplification for PQQ detection down to the picomolar levels is well-predicted. These performances outperform by several orders of magnitude the direct electrochemical detection of PQQ in solution and by 1 to 2 orders the detection limits previously achieved by UV-vis spectroscopic detection of the homogeneous PQQ-GDH reconstitution.
Assuntos
Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Enzimas Imobilizadas/química , Glucose Desidrogenase/química , Animais , Bovinos , EletrodosRESUMO
Mitochondrial F(1)F(o) ATP synthase is an enzymatic complex involved in the aerobic synthesis of ATP. It is well known that several enzymes are organized in supramolecular complexes in the inner mitochondrial membrane. The ATP synthase supramolecular assembly is mediated through two interfaces. One leads to dimer formation and the other to oligomer formation. In yeast, the presence of ATP synthase oligomers has been described as essential to the maintenance of the mitochondrial cristae ultrastructure. Indeed, the destabilization of the interactions between monomers was shown to alter the organization of the inner mitochondrial membrane, leading to the formation of onion-like structures similar to those observed in some mitochondrial pathologies. By using information obtained this decade (structure modeling, electron microscopy and cross-linking), this paper (i) reviews the actual state of the art and (ii) proposes a topological model of the transmembrane domains and interfaces of the ATP synthase's tetramer. This review also discusses the physiological role of this oligomerization process and its potential implications in mammal pathology. This article is part of a Directed Issue entitled: Bioenergetic Dysfunction, adaptation and therapy.
Assuntos
Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Dimerização , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Moleculares , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The involvement of subunit 6 (a) in the interface between yeast ATP synthase monomers has been highlighted. Based on the formation of a disulfide bond and using the unique cysteine 23 as target, we show that two subunits 6 are close in the inner mitochondrial membrane and in the solubilized supramolecular forms of the yeast ATP synthase. In a null mutant devoid of supernumerary subunits e and g that are involved in the stabilization of ATP synthase dimers, ATP synthase monomers are close enough in the inner mitochondrial membrane to make a disulfide bridge between their subunits 6, and this proximity is maintained in detergent extract containing this enzyme. The cross-linking of cysteine 23 located in the N-terminal part of the first transmembrane helix of subunit 6 suggests that this membrane-spanning segment is in contact with its counterpart belonging to the ATP synthase monomer that faces it and participates in the monomer-monomer interface.
Assuntos
Membranas Mitocondriais/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Multimerização Proteica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Methylmalonate-semialdehyde dehydrogenase (MSDH) belongs to the CoA-dependent aldehyde dehydrogenase subfamily. It catalyzes the NAD-dependent oxidation of methylmalonate semialdehyde (MMSA) to propionyl-CoA via the acylation and deacylation steps. MSDH is the only member of the aldehyde dehydrogenase superfamily that catalyzes a ß-decarboxylation process in the deacylation step. Recently, we demonstrated that the ß-decarboxylation is rate-limiting and occurs before CoA attack on the thiopropionyl enzyme intermediate. Thus, this prevented determination of the transthioesterification kinetic parameters. Here, we have addressed two key aspects of the mechanism as follows: 1) the molecular basis for recognition of the carboxylate of MMSA; and 2) how CoA binding modulates its reactivity. We substituted two invariant arginines, Arg-124 and Arg-301, by Leu. The second-order rate constant for the acylation step for both mutants was decreased by at least 50-fold, indicating that both arginines are essential for efficient MMSA binding through interactions with the carboxylate group. To gain insight into the transthioesterification, we substituted MMSA with propionaldehyde, as both substrates lead to the same thiopropionyl enzyme intermediate. This allowed us to show the following: 1) the pK(app) of CoA decreases by â¼3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 10(4)-fold relative to a chemical model. Moreover, we observed binding of CoA to the acylation complex, supporting a CoA-binding site distinct from that of NAD(H).
Assuntos
Bacillus subtilis/enzimologia , Coenzima A/metabolismo , Metilmalonato-Semialdeído Desidrogenase (Acilante)/metabolismo , Aldeídos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Biocatálise , Estabilidade Enzimática , Esterificação , Humanos , Cinética , Metilmalonato-Semialdeído Desidrogenase (Acilante)/química , Metilmalonato-Semialdeído Desidrogenase (Acilante)/genética , Ácido Metilmalônico/química , Ácido Metilmalônico/metabolismo , Dados de Sequência Molecular , Mutação , NAD/metabolismo , Ligação Proteica , Ratos , Especificidade por SubstratoRESUMO
We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.
Assuntos
Acinetobacter calcoaceticus/enzimologia , Fontes de Energia Bioelétrica , Técnicas Biossensoriais , Glucose Desidrogenase/química , Glucose/análise , Glucose Desidrogenase/genética , Mutagênese , Mutação , Conformação Proteica , Engenharia de ProteínasRESUMO
Over the past 15 years, mechanistic and structural aspects were studied extensively for hydrolytic ALDHs. One the most striking feature of nearly all X-ray structures of binary ALDH-NAD(P)(+) complexes is the great conformational flexibility of the NMN moiety of the NAD(P)(+), in particular of the nicotinamide ring. However, the fact that the acylation step is efficient in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans and in other hydrolytic ALDHs implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary complex to allow an efficient and stereospecific hydride transfer. Another key aspect of the chemical mechanism of this ALDH family is the requirement for the reduced NMN (NMNH) to move away from the initial position of the NMN for adequate positioning and activation of the deacylating water molecule by invariant E268 for completion of the reaction. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the stabilization of the NMN moiety of the cofactor during the acylation step and to provide structural evidence of conformational isomerization of the cofactor during the catalytic cycle of hydrolytic ALDHs. The results presented here will be discussed for their relevance to the two-step catalytic mechanism and from an evolutionary viewpoint.
Assuntos
Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/química , Biocatálise , Estabilidade Enzimática , Hidrólise , Isomerismo , Modelos Moleculares , Niacinamida/química , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
Homotetrameric MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis catalyses the NAD-dependent oxidation of MMSA (methylmalonate semialdehyde) and MSA (malonate semialdehyde) into PPCoA (propionyl-CoA) and acetyl-CoA respectively via a two-step mechanism. In the present study, a detailed mechanistic characterization of the MSDH-catalysed reaction has been carried out. The results suggest that NAD binding elicits a structural imprinting of the apoenzyme, which explains the marked lag-phase observed in the activity assay. The enzyme also exhibits a half-of-the-sites reactivity, with two subunits being active per tetramer. This result correlates well with the presence of two populations of catalytic Cys302 in both the apo- and holo-enzymes. Binding of NAD causes a decrease in reactivity of the two Cys302 residues belonging to the two active subunits and a pKapp shift from approx. 8.8 to 8.0. A study of the rate of acylation as a function of pH revealed a decrease in the pKapp of the two active Cys302 residues to approx. 5.5. Taken to-gether, these results support a sequential Cys302 activation process with a pKapp shift from approx. 8.8 in the apo-form to 8.0 in the binary complex and finally to approx. 5.5 in the ternary complex. The rate-limiting step is associated with the b-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before transthioesterification. These data also indicate that bicarbonate, the formation of which is enzyme-catalysed, is the end-product of the reaction.
Assuntos
Bacillus subtilis/enzimologia , Metilmalonato-Semialdeído Desidrogenase (Acilante)/metabolismo , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Apoenzimas/metabolismo , Bicarbonatos , Catálise , Cisteína/metabolismo , Descarboxilação , Dissulfetos/química , Holoenzimas/metabolismo , Concentração de Íons de Hidrogênio , Iodoacetamida/química , Cinética , Mutação/genética , NAD/metabolismo , Oxirredução , Ligação Proteica , Fatores de TempoRESUMO
Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Suitable crystals for X-ray diffraction experiments were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 195.2, b = 192.5, c = 83.5 A, and contain one tetramer per asymmetric unit. X-ray diffraction data were collected to 2.5 A resolution using a synchrotron-radiation source. The crystal structure was solved by the molecular-replacement method.