Formation of adenosine 5′-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli

AbstractThe mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5′-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes

Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes

International audienceAfter enzymes were first discovered in the late XIX century, and for the first seventy years of enzymology, kinetic experiments were the only source of information about enzyme mechanisms. Over the following fifty years, these studies were taken over by approaches that give information at the molecular level, such as crystallography, spectroscopy and theoretical chemistry (as emphasized by the Nobel Prize in Chemistry awarded last year to M. Karplus, M. Levitt and A. Warshel). In this review, we thoroughly discuss the interplay between the information obtained from theoretical and experimental methods, by focussing on enzymes that process small molecules such as H 2 or CO 2 (hydrogenases, CO-dehydrogenase and carbonic anhydrase), and that are therefore relevant in the context of energy and environment. We argue that combining theoretical chemistry (DFT, MD, QM/MM) and detailed investigations that make use of modern kinetic methods, such as protein film voltammetry, is an innovative way of learning about individual steps and/or complex reactions that are part of the catalytic cycles. We illustrate this with recent results from our labs and others, including studies of gas transport along substrate channels, long range proton transfer, and mechanisms of catalysis, inhibition or inactivation. Broader context Some reactions which are very important in the context of energy and environment, such as the conversion between CO and CO2 , or H+ and H2 , are catalyzed in living organisms by large and complex enzymes that use inorganic active sites to transform substrates, chains of redox centers to transfer electrons, ionizable amino acids to transfer protons, and networks of hydrophobic cavities to guide the diffusion of substrates and products within the protein. This highly sophisticated biological plumbing and wiring makes turnover frequencies of thousands of substrate molecules per second possible. Understanding the molecular details of catalysis is still a challenge. We explain in this review how a great deal of information can be obtained using an interdisciplinary approach that combines state-of-the art kinetics and computational chemistry. This differs from—and complements—the more traditional strategies that consist in trying to see the catalytic intermediates using methods that rely on the interaction between light and matter, such as X-ray diffraction and spectroscopic techniques

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

13. Structure et mécanisme des hydrogénases

La production d’énergie dans les êtres vivants fait intervenir des enzymes qui utilisent soit l’énergie lumineuse, soit l’énergie de certaines réactions chimiques d’oxydoréduction* (réactions d’échange d’électrons*) pour synthétiser de l’ATP* (cf. II.8). Le dihydrogène est l’un des combustibles biologiques oxydés dans ces réactions. Il joue par ailleurs un rôle important dans le métabolisme de la plupart des bactéries et de certaines algues unicellulaires. Certains micro-organismes le produis..

Visualizing the gas channel of a monofunctional carbon monoxide dehydrogenase

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L’électrochimie, un outil pour étudier les mécanismes enzymatiques

International audienceLe fonctionnement des enzymes qui catalysent des réactions redox fait intervenir des étapes très diverses (diffusion du substrat à l’intérieur de l’enzyme, réactions chimiques au site actif, transferts à longue distance d’électrons et de protons) et qui impliquent des sites de la protéine distants les uns des autres.Cet article illustre, en prenant pour exemple les enzymes qui catalysent l’oxydation réversible du dihydrogène, comment l’électrochimie peut maintenant être utilisée en combinaison avec d’autres approches comme la chimie théorique et la mutagenèse dirigée, pour étudier des aspects variés du mécanisme moléculaire des enzymes redox

L’électrochimie, un outil pour étudier les mécanismes enzymatiques

International audienceLe fonctionnement des enzymes qui catalysent des réactions redox fait intervenir des étapes très diverses (diffusion du substrat à l’intérieur de l’enzyme, réactions chimiques au site actif, transferts à longue distance d’électrons et de protons) et qui impliquent des sites de la protéine distants les uns des autres.Cet article illustre, en prenant pour exemple les enzymes qui catalysent l’oxydation réversible du dihydrogène, comment l’électrochimie peut maintenant être utilisée en combinaison avec d’autres approches comme la chimie théorique et la mutagenèse dirigée, pour étudier des aspects variés du mécanisme moléculaire des enzymes redox

Towards engineering O 2 -tolerance in [Ni–Fe] hydrogenases

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Mechanism of inhibition of NiFe hydrogenase by nitric oxide

International audienceHydrogenases reversibly catalyze the oxidation of molecular hydrogen and are inhibited by several small molecules including O2, CO and NO. In the present work, we investigate the mechanism of inhibition by NO of the oxygen-sensitive NiFe hydrogenase from Desulfovibrio fructosovorans by coupling site-directed mutagenesis, protein film voltammetry (PFV) and EPR spectroscopy. We show that micromolar NO strongly inhibits NiFe hydrogenase and that the mechanism of inhibition is complex, with NO targeting several metallic sites in the protein. NO reacts readily at the NiFe active site according to a two-step mechanism. The first and faster step is the reversible binding of NO to the active site followed by a slower and irreversible transformation at the active site. NO also induces irreversible damage of the iron–sulfur centers chain. We give direct evidence of preferential nitrosylation of the medial [3Fe–4S] to form dinitrosyl–iron complexes