Mechanism of reconstitution/activation of the soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus: a comprehensive study

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Caractérisation enzymatique de la Méthylmalonate semialdéhyde déshydrogénase de Bacillus subtilis

Homotetrameric methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis catalyzes the NAD+-dependent oxidation of methylmalonate semialdehyde into propionyl-Coenzyme A via a two-step mechanism. A detailed mechanistic characterization of the MSDH-catalyzed reaction has been carried out. The results show that NAD binding elicits a structural imprinting of the apo-enzyme. The enzyme exhibits a half-of-the-site reactivity with two subunits active per tetramer. The results support also a sequential C302 activation process with a pKapp shift from ~ 8.8 in the apo-form to 8.0 in the binary complex and finally ~ 5.5 in the ternary complex. The rate-limiting step is shown to be associated with the beta-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before the transthioesterification step. Binding of the substrate is favoured by stabilizing interactions between its carboxylate group and the invariant residues R124 and R301.La méthylmalonate semialdéhyde déshydrogénase (MSDH) de Bacillus subtilis, enzyme homotétramérique, catalyse l'oxydation NAD+-dépendante du méthylmalonate semialdéhyde en propionyl-coenzyme A via un mécanisme à deux étapes. Le mécanisme de la réaction a été étudié. Les résultats montrent que la liaison du NAD entraîne un réarrangement local conformationnel de la MSDH et que la MSDH présente une réactivité de demi-site avec deux sous-unités actives par tétramère. Les résultats montrent également un mécanisme séquentiel d'activation de la Cys 302 catalytique, son pKapp passant de 8.8 dans l'apoenzyme à 8 dans le complexe binaire et enfin à 5.5 dans le complexe ternaire. L'étape limitante de la réaction est associée au processus de beta-décarboxylation qui a lieu après le relargage du NADH du complexe ternaire thioacylenzyme et avant l'étape de transthioestérification. La fixation du substrat est stabilisée par l'interaction de son carboxylate avec les résidus invariants R124 et R301

Caractérisation enzymatique de la Méthylmalonate semialdéhyde déshydrogénase de Bacillus subtilis

La méthylmalonate semialdéhyde déshydrogénase (MSDH) de Bacillus subtilis, enzyme homotétramérique, catalyse l'oxydation NAD+-dépendante du méthylmalonate semialdéhyde en propionyl-coenzyme A via un mécanisme à deux étapes. Le mécanisme de la réaction a été étudié. Les résultats montrent que la liaison du NAD entraîne un réarrangement local conformationnel de la MSDH et que la MSDH présente une réactivité de demi-site avec deux sous-unités actives par tétramère. Les résultats montrent également un mécanisme séquentiel d'activation de la Cys 302 catalytique, son pKapp passant de 8.8 dans l'apoenzyme à 8 dans le complexe binaire et enfin à 5.5 dans le complexe ternaire. L'étape limitante de la réaction est associée au processus de beta-décarboxylation qui a lieu après le relargage du NADH du complexe ternaire thioacylenzyme et avant l'étape de transthioestérification. La fixation du substrat est stabilisée par l'interaction de son carboxylate avec les résidus invariants R124 et R301.Homotetrameric methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis catalyzes the NAD+-dependent oxidation of methylmalonate semialdehyde into propionyl-Coenzyme A via a two-step mechanism. A detailed mechanistic characterization of the MSDH-catalyzed reaction has been carried out. The results show that NAD binding elicits a structural imprinting of the apo-enzyme. The enzyme exhibits a half-of-the-site reactivity with two subunits active per tetramer. The results support also a sequential C302 activation process with a pKapp shift from ~ 8.8 in the apo-form to 8.0 in the binary complex and finally ~ 5.5 in the ternary complex. The rate-limiting step is shown to be associated with the beta-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before the transthioesterification step. Binding of the substrate is favoured by stabilizing interactions between its carboxylate group and the invariant residues R124 and R301.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Incorporating clickers into an enzymology course improves student performance

International audienceHere, we describe how poor exam results of undergraduate students enrolled in an enzymology course at the University of Bordeaux wereimproved through the introduction of ‘clickers’ as an audience response system. By using clickers only in a small-group tutorial element of a largetheoretical course, we observed an improvement in exam scores that resulted in a lower failure rate for the course. Furthermore, students of allabilities were found to benefit from their use. Students reported better retention of both lecture and tutorial content. An analysis of how clickerswere employed within the tutorials indicated that the use of clickers to promote discussion and impart knowledge likely resulted in a moderateimprovement of exam scores. We hypothesize that students were more prepared for exams through greater reflection of exam questions, resulting inan enhanced ability to retrieve memorized information and apply it within a time-limited exam setting

Designing a highly active soluble PQQ–glucose dehydrogenase for efficient glucose biosensors and biofuel cells

5 pagesInternational audienceWe 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

An enzymatic glucose/O2 biofuel cell operating in human blood

Enzymatic biofuel cells (BFCs) may power implanted medical devices and will rely on the use of glucoseand O2 available in human bodily fluids. Other than well-established experiments in aqueous buffer, littlework has been performed in whole human blood because it contains numerous inhibiting molecules....Ingénierie de matériaux poreux et d'enzymes pour le développement de biopilesInitiative d'excellence de l'Université de Bordeau

Switching an O2 sensitive glucose oxidase bioelectrode into an almost insensitive one by cofactor redesign

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

Bilirubin oxidase-based silica macrocellular robust catalyst for on line dyes degradation

International audienceWe 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 (RRBR) at two different pH (4 and 8.2), both in presence or absence of redox mediator. 100% decolorization efficiency was reached within 8 hours at pH 4 for Congo Red without the need of a redox mediator. 80% efficiency was reached for RBBR at pH 8.2 in 24 hours without redox mediator still. 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

Heterogeneous Reconstitution of the PQQ-Dependent Glucose Dehydrogenase Immobilized on an Electrode: A Sensitive Strategy for PQQ Detection Down to Picomolar Levels

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...

Methylmalonate-semialdehyde Dehydrogenase from Bacillus subtilis: SUBSTRATE SPECIFICITY AND COENZYME A BINDING*

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 β-decarboxylation process in the deacylation step. Recently, we demonstrated that the β-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 pKapp of CoA decreases by ∼3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 104-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)