Eric Chabriere

Crystal structures of the key anaerobic enzyme pyruvate:ferredoxin oxidoreductase, free and in complex with pyruvate

Oxidative decarboxylation of pyruvate to form acetyl–coenzyme A, a crucial step in many metabolic pathways, is carried out in most aerobic organisms by the multienzyme complex pyruvate dehydrogenase. In most anaerobes, the same reaction is usually catalyzed by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFOR). Thus, PFOR is a potential target for drug design against certain anaerobic pathogens. Here, we report the crystal structures of the homodimeric Desulfovibrio africanus PFOR (data to 2.3 Å resolution), and of its complex with pyruvate (3.0 Å resolution). The structures show that each subunit consists of seven domains, one of which affords protection against oxygen. The thiamin pyrophosphate (TPP) cofactor and the three [4Fe–4S] clusters are suitably arranged to provide a plausible electron transfer pathway. In addition, the PFOR–pyruvate complex structure shows the noncovalent fixation of the substrate before the catalytic reaction.

The molecular basis of phosphate discrimination in arsenate-rich environments.

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pKa values, similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches. However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter’s presence therefore decouples metabolism. Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-ångström-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole–anion interactions, and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 103 excess) even in highly arsenate-rich environments.

Structural basis for natural lactonase and promiscuous phosphotriesterase activities.

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 A resolution) and in complex with a quorum-sensing lactone mimic at 2.0 A resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.

Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates

BACKGROUND Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. METHODS Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. RESULTS Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. CONCLUSIONS NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

Characterisation of the organophosphate hydrolase catalytic activity of SsoPox

SsoPox is a lactonase endowed with promiscuous phosphotriesterase activity isolated from Sulfolobus solfataricus that belongs to the Phosphotriesterase-Like Lactonase family. Because of its intrinsic thermal stability, SsoPox is seen as an appealing candidate as a bioscavenger for organophosphorus compounds. A comprehensive kinetic characterisation of SsoPox has been performed with various phosphotriesters (insecticides) and phosphodiesters (nerve agent analogues) as substrates. We show that SsoPox is active for a broad range of OPs and remains active under denaturing conditions. In addition, its OP hydrolase activity is highly stimulated by anionic detergent at ambient temperature and exhibits catalytic efficiencies as high as kcat/KM of 105 M−1s−1 against a nerve agent analogue. The structure of SsoPox bound to the phosphotriester fensulfothion reveals an unexpected and non-productive binding mode. This feature suggests that SsoPoxs active site is sub-optimal for phosphotriester binding, which depends not only upon shape but also on localised charge of the ligand.

Structure and electron transfer mechanism of pyruvate:ferredoxin oxidoreductase

The first crystal structure of pyruvate:ferredoxin oxidoreductase to be determined has provided significant new information on its structural organization and redox chemistry. Spectroscopic analyses of a radical reaction intermediate have shed more light on its thiamin-based mechanism of catalysis. Different approaches have been used to study the interaction between the enzyme and ferredoxin, its redox partner.

Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus

Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading OPs are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (PTE) (SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against “quorum sensing signals”, and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic PTEs from Pseudomonas diminuta and Agrobacterium radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers.

Structural and Enzymatic characterization of the lactonase SisLac from Sulfolobus islandicus

Background A new member of the Phosphotriesterase-Like Lactonases (PLL) family from the hyperthermophilic archeon Sulfolobus islandicus (SisLac) has been characterized. SisLac is a native lactonase that exhibits a high promiscuous phosphotriesterase activity. SisLac thus represents a promising target for engineering studies, exhibiting both detoxification and bacterial quorum quenching abilities, including human pathogens such as Pseudomonas aeruginosa. Methodology/Principal Findings Here, we describe the substrate specificity of SisLac, providing extensive kinetic studies performed with various phosphotriesters, esters, N-acyl-homoserine lactones (AHLs) and other lactones as substrates. Moreover, we solved the X-ray structure of SisLac and structural comparisons with the closely related SsoPox structure highlighted differences in the surface salt bridge network and the dimerization interface. SisLac and SsoPox being close homologues (91% sequence identity), we undertook a mutational study to decipher these structural differences and their putative consequences on the stability and the catalytic properties of these proteins. Conclusions/Significance We show that SisLac is a very proficient lactonase against aroma lactones and AHLs as substrates. Hence, data herein emphasize the potential role of SisLac as quorum quenching agent in Sulfolobus. Moreover, despite the very high sequence homology with SsoPox, we highlight key epistatic substitutions that influence the enzyme stability and activity.

Elucidation of the phosphate binding mode of DING proteins revealed by subangstrom X-ray crystallography.

PfluDING is a bacterial protein isolated from Pseudomonas fluorescens that belongs to the DING protein family, which is ubiquitous in eukaryotes and extends to prokaryotes. DING proteins and PfluDING have very similar topologies to phosphate Solute Binding Proteins (SBPs). The three-dimensional structure of PfluDING was obtained at subangstrom resolution (0.88 and 0.98 A) at two different pHs (4.5 and 8.5), allowing us to discuss the hydrogen bond network that sequesters the phosphate ion in the binding site. From this high resolution data, we experimentally elucidated the molecular basis of phosphate binding in phosphate SBPs. The phosphate ion is tightly bound to the protein via 12 hydrogen bonds between phosphate oxygen atoms and OH and NH groups of the protein. The proton on one oxygen atom of the phosphate dianion forms a 2.5 A low barrier hydrogen bond with an aspartate, with the energy released by forming this strong bond ensuring the specificity for the dianion even at pH 4.5. In particular, contrary to previous theories on phosphate SBPs, accurate electrostatic potential calculations show that the binding cleft is positively charged. PfluDING structures reveal that only dibasic phosphate binds to the protein at both acidic and basic phosphate, suggesting that the protein binding site environment stabilizes the HPO(4)(2-) form of phosphate.

Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox.

Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.