Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms.... more Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms. The thermoalkaliphile Caldalkalibacillus thermarum TA .A is an organism of interest for studies into how alkaliphiles adapt to their extreme lifestyles, as it can grow from pH. to pH. Within most classes of microbes, the membrane-bound electron transport chain (ETC) enables a great degree of adaptability and is a key part of metabolic adaptation. Knowing what membrane proteins are generally expressed is crucial as a benchmark for further studies. Unfortunately, membrane proteins are the category of proteins hardest to detect using conventional cellular proteomics protocols. In part, this is due to the hydrophobicity of membrane proteins as well as their general lower absolute abundance, which hinders detection. Here, we performed a combination of whole cell lysate proteomics and proteomics of membrane extracts solubilised with either SDS or FOS-choline-at various temperatures. The combined methods led to the detection of membrane proteins containing at least a single transmembrane helix (TMH). Within this data set we revealed a full oxidative phosphorylation pathway as well as an alternative NADH dehydrogenase type II (Ndh-) and a microaerophilic cytochrome oxidase ba. We also observed C. thermarum TA .A expressing transporters for ectoine and glycine betaine, compounds that are known osmolytes that may assist in maintaining a near neutral internal pH when the external pH is highly alkaline.
Applied and Environmental Microbiology, Oct 15, 2010
The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknow... more The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknown. We demonstrate that alkaliphilic (pH > 9) Bacillus species are sensitive to artificial iron (Fe 3؉) chelators and produce iron-chelating molecules. These alkaliphilic siderophores contain catechol and hydroxamate moieties, and their synthesis is stimulated by manganese(II) salts and suppressed by FeCl 3 addition. Purification and mass spectrometric characterization of the siderophore produced by Caldalkalibacillus thermarum failed to identify any matches to previously observed fragmentation spectra of known siderophores, suggesting a novel structure.
It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F 1 F o ATP syntha... more It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F 1 F o ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F 1 F o ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F 1 F o ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg 2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F 1 F o ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg 2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by secondshell residues thus the function of the ε subunit changes with growth conditions.
ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found i... more ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found in living cells. These enzymes are driven by an electrochemical ion gradient, which allows the catalytic evolution of ATP by a binding change mechanism. Most ATP synthases are capable of catalysing ATP hydrolysis to varying degrees, and to prevent wasteful ATP hydrolysis, bacteria and mitochondria have regulatory mechanisms such as ADP inhibition. Additionally, 1 subunit inhibition has also been described in three bacterial systems, Escherichia coli, Bacillus PS3 and Caldalkalibacillus thermarum TA2.A1. Previous studies suggest that the 1 subunit is capable of undergoing an ATP-dependent conformational change from the ATP hydrolytic inhibitory 'extended' conformation to the ATP-induced non-inhibitory 'hairpin' conformation. A recently published crystal structure of the F 1 domain of the C. thermarum TA2.A1 F 1 F o ATP synthase revealed a mutant 1 subunit lacking the ability to bind ATP in a hairpin conformation. This is a surprising observation considering it is an organism that performs no ATP hydrolysis in vivo, and appears to challenge the current dogma on the regulatory role of the 1 subunit. This has prompted a re-examination of present knowledge of the 1 subunits role in different organisms. Here, we compare published biochemical, biophysical and structural data involving 1 subunitmediated ATP hydrolysis regulation in a variety of organisms, concluding that the 1 subunit from the bacterial F-type ATP synthases is indeed capable of regulating ATP hydrolysis activity in a wide variety of bacteria, making it a potentially valuable drug target, but its exact role is still under debate.
Biochimica Et Biophysica Acta - Bioenergetics, Sep 1, 2018
a mutation in this gene (m.8969GNA), leading to replacement of a highly conserved serine residue ... more a mutation in this gene (m.8969GNA), leading to replacement of a highly conserved serine residue into asparagine at amino acid position 148, in a 14-year-old Chinese female who initially developed an isolated nephropathy followed by a complex clinical presentation with brain and muscle problems [1]. An equivalent of this mutation in yeast (atp6-S175N) was shown to prevent F O-mediated proton transfer and weaken F O to F 1 association. Herein we identified four first-site intragenic suppressors (aN 175 D, aN 175 K, aN 175 I, and aN 175 T), which, in light of a recently published atomic structure of yeast F O , provides strong indication that the detrimental consequences of the original mutation result from the establishment of a hydrogen bound between aN 175 and a nearby glutamate residue (aE 172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS 175 N mutation can be suppressed by second-site suppressors (aP 12 S, aI 171 F, aI 171 N, aI 239 F, and aI 200 M), of which some are very distantly located (by 20-30 Å) from the original mutation. The possibility to disrupt a pathogenic hydrogen bond through long-range effects is an interesting observation that holds promise for the development of therapeutic molecules.
Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of... more Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent K M value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo 3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo 3. Cardiolipin (CL) is an anionic phospholipid that consists of two phosphatidyl groups connected by a glycerol moiety. CL is important for optimal function of various eukaryotic and prokaryotic membrane protein complexes 1-5. CL can interact with bacterial respiratory complexes from phylogenetically diverse species, such as Mycobacterium phlei 6 , Rhodobacter sphaeroides 7 and Escherichia coli 8. Among E. coli respiratory chain complexes, CL was shown to activate purified, detergent-free cytochrome bo 3 9 and was the most efficient phospholipid for activation of detergent-solubilized NADH dehydrogenase 10 and of liposome-reconstituted nitrate reductase 11. Defined binding sites for CL have been determined in crystal structures of E. coli formate dehydrogenase N 12 , succinate dehydrogenase 13 , and nitrate reductase 11. The respiratory chain in Escherichia coli features a heme-copper-type terminal oxidase, cytochrome bo 3 , which transfers electrons from quinol-type substrates onto molecular oxygen. Next to this energetically efficient terminal oxidase, E. coli utilizes cytochrome bd as an alternative branch of the respiratory chain. Cytochrome bd oxidizes quinols, like ubiquinol or menaquinol, coupled with reduction of molecular oxygen to water (Fig. 1A) 14,15. Cytochrome bd is particularly important under conditions of stress, such as O 2-limitation 16 , in the presence of nitric oxide 17,18 hydrogen peroxide 19-21 and hydrogen sulfide 22,23. Lack of cytochrome bd in uropathogenic E. coli strains led to attenuation in mouse infection models 24. Cytochrome bd is present in of a broad variety of Gram-positive and Gram-negative bacteria and archea, but not in the respiratory chain of eukaryotes 14. Purified active cytochrome bd has been prepared from several bacterial species, including E. coli 25,26 , Azotobacter vinelandii 27 , Corynebacterium glutamicum 28 and Geobacillus thermodenitrificans 29,30. However, to our knowledge there are no data available concerning the effect of CL on cytochrome bd activity. In this report, we investigated the influence of CL on detergent-purified cytochrome bd. We found that CL activated the enzymatic activity of cytochrome bd from E. coli, G. thermodenitrificans and C. glutamicum. We then extended our experimentation and also assessed the impact of CL on the activity of purified cytochrome bo 3 from E. coli.
Abstract The multiwall carbon nanotubes-polypyrrole (MWCNTs-PPy) composite is a well-known hybrid... more Abstract The multiwall carbon nanotubes-polypyrrole (MWCNTs-PPy) composite is a well-known hybrid material suitable for energy storage applications such as capacitors. Since the electrochemical activity, and long-term stability of carbon-PPy systems are critical for their application, we examined the effect of grafting PPy via a 3-ABA linker with MWCNTs with respect to the preference of the donor–acceptor pairs, and furthermore, the durability. X-ray photoelectron spectroscopy studies of C 1s and O 1s signals reveal the electronic interaction between carbon and polymer (π−π* stacking), as well as a binding energy shift for C 1s and O 1s carbonyl signals due to the chemical bonding via the electron donating linker and grafted polymer. Cyclic voltammetry and galvanostatic charge–discharge studies of the capacitor electrode reveal the effect of the reversal doping of the polymer with chloride, as well as the synergy of double layer capacitance (MWCNTs) and pseudo-capacitance of the poly(pyrrole). Impedance spectroscopy studies confirm the improved electrochemical stability for the composite (stable until 1.2 V) in comparison to the bare polymer (degradation at 0.58 V).
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The ε subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevaili... more The ε subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevailing evidence supports the notion that when the ATP concentration falls below a certain threshold, the ε subunit changes its conformation from a non-inhibitory down-state to an extended up-state which then inhibits enzymatic ATP hydrolysis by binding to the catalytic domain. It has been demonstrated that the ε subunit from Bacillus PS3 is selective for ATP over other nucleotides, including GTP. In this study, the purine triphosphate selectivity is rationalized via results from MD simulations and free energy calculations for the R103A/R115A mutant of the ε subunit from Bacillus PS3, which binds ATP stronger than the wild-type (WT) protein. Our results are in good agreement with experimental data, and the elucidated molecular basis for selectivity could help to guide the design of novel GTP sensors.
Proceedings of the National Academy of Sciences, 2020
Significance F-ATP synthase is a fundamental enzyme supplying adenosine triphosphate (ATP), sprea... more Significance F-ATP synthase is a fundamental enzyme supplying adenosine triphosphate (ATP), spreading across all kingdoms of life. Despite remarkable conservation of its basic structure and function, biophysical studies have revealed discrete differences in the rotary mechanisms of bacterial and eukaryotic F 1 -ATPases (the catalytic portions of the enzymes). Here, we analyzed the rotational dynamics of Paracoccus denitrificans F 1 (PdF 1 ), a bacterial F 1 -ATPase that exhibits high homology with the core functional subunits of its mitochondrial counterpart. Notably, PdF 1 possesses a simplified chemomechanical scheme different from that of all other F 1 -ATPases. Our results reveal an unexpected diversity in the chemomechanical coupling cycle of the F 1 -ATPase machinery and show that features such as homology or phylogenetic relationship cannot uniquely define the rotary scheme pattern.
Diastereomers are characterised by an intrinsic energy difference, and thermodynamics dictate the... more Diastereomers are characterised by an intrinsic energy difference, and thermodynamics dictate their distribution within a dynamic equilibrium. The characteristic mechanistic reversibility and non‐ideal stereoselectivity of catalysts therefore simultaneously promote both synthesis and epimerization of products during the formation of diastereomers. This feature can even result in the thermodynamic inversion of a chiral centre against the catalyst's stereoselectivity. Here, we provide a comprehensive experimental and theoretical study of factors that govern thermodynamic epimerization in catalysis, using enzymes as example. Our analysis highlights, that the deduction of a catalyst's stereoselectivity based on the absolute configuration of the isolated product constitutes a potential pitfall. The selective formation of either the thermodynamic‐, or the kinetic product is less determined by the catalyst, but rather by the reaction conditions. Next to low temperatures, a high max...
A novel large-scale survey approach for microbial nonulosonic acids (sialic acids) including a fi... more A novel large-scale survey approach for microbial nonulosonic acids (sialic acids) including a first molecular level comparative study is presented.
The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP),... more The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can trans...
The digestive enzyme papain can drive the formation of crystalline titanium dioxide nanoparticles... more The digestive enzyme papain can drive the formation of crystalline titanium dioxide nanoparticles on surfaces. This dual functionality of thin film formation and mineralization promotion has the potential to enable the construction of hierarchical inorganic/organic structures in the form of continuous amorphous titania/protein films.
The aerobic thermoalkaliphile Caldalkalibacillus thermarum strain TA2.A1 is a member of a separat... more The aerobic thermoalkaliphile Caldalkalibacillus thermarum strain TA2.A1 is a member of a separate order of alkaliphilic bacteria closely related to the Bacillales order. Efforts to relate the genomic information of this evolutionary ancient organism to environmental adaptation have been thwarted by the inability to construct a complete genome. The existing draft genome is highly fragmented due to repetitive regions, and gaps between and over repetitive regions were unbridgeable. To address this, Oxford Nanopore Technology’s MinION allowed us to span these repeats through long reads, with over 6000-fold coverage. This resulted in a single 3.34 Mb circular chromosome. The profile of transporters and central metabolism gives insight into why the organism prefers glutamate over sucrose as carbon source. We propose that the deamination of glutamate allows alkalization of the immediate environment, an excellent example of how an extremophile modulates environmental conditions to suit its...
The class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hyd... more The class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hydroxypyruvate as nucleophilic donor substrate, giving access to synthetically challenging 3,4dihydroxy-α-ketoacids. The crystal structure of holo-SwHKA in complex with hydroxypyruvate revealed CH-π interactions between the CÀ H bonds at C3 of hydroxypyruvate and a phenylalanine residue at position 210, which in this case occupies the position of a conserved leucine residue. Mutagenesis to tyrosine further increased the electron density of the interacting aromatic system and effected a rate enhancement by twofold. While the leucine variant efficiently catalyses the enolisation of hydroxypyruvate as the first step in the aldol reaction, the enol intermediate then becomes trapped in a disfavoured configuration that considerably hinders subsequent CÀ C bond formation. In SwHKA, micromolar concentrations of inorganic phosphate increase the catalytic rate constant of enolisation by two orders of magnitude. This rate enhancement was now shown to be functionally conserved across the structurally distinct (α/β) 8 barrel and αββα sandwich folds of two pyruvate aldolases. Characterisation of the manganese (II) cofactor by electron paramagnetic resonance excluded ionic interactions between the metal centre and phosphate. Instead, histidine 44 was shown to be primarily responsible for the binding of phosphate in the micromolar range and the observed rate enhancement in SwHKA.
Materials for Renewable and Sustainable Energy, 2015
Nitrogen-doped carbon is a promising metalfree catalyst for oxygen reduction reaction in fuel cel... more Nitrogen-doped carbon is a promising metalfree catalyst for oxygen reduction reaction in fuel cells and metal-air batteries. However, its practical application necessitates a significant cost reduction, which can be achieved in part by using new synthetic methods and improvement of catalytic activity by increasing the density of redox active centers. This can be modulated by using polymer as the carbon and nitrogen sources. Although, superior catalytic activity of such N-doped C has been investigated in details, the electrochemical long-term stability of polymer-derived doped-carbon is still unclear. Herein, in this study we generated N-doped carbon from the most recommended polymer that is comparable to the state-of-the-art materials with porosity as high as 2,086 m 2 g-1 and a nitrogen doping level of 3-4 at.%, of which 56 % is pyrrolic N, 36.1 % pyridinic and *8 % graphitic. The electrochemical characterization shows that N-doped carbon is catalytic toward oxygen reduction in an alkaline electrolyte via a favorable four-electron process, however, not stable under long-term potential scanning. The irreversible electrochemical oxidation of this material is associated with the presence of a significant content or pyrrolic and pyridinic N close to the edge of the carbon network originating from the polypyrrole precursor. These structures are less stable under operating electrochemical potential. The role of polypyrrole as the precursor of N-doped carbons has to be carefully revised since it supplies sufficient number of catalytic sites, but also generates unstable functionalities on the carbon surface.
Protein-protein interactions are well-known to regulate enzyme activity in cell signaling and met... more Protein-protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein-protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis.W e report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis. Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c 3 (Fcc 3 ) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc 3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc 3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein-protein interactions.
Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the mol... more Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged Cterminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.
Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms.... more Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms. The thermoalkaliphile Caldalkalibacillus thermarum TA .A is an organism of interest for studies into how alkaliphiles adapt to their extreme lifestyles, as it can grow from pH. to pH. Within most classes of microbes, the membrane-bound electron transport chain (ETC) enables a great degree of adaptability and is a key part of metabolic adaptation. Knowing what membrane proteins are generally expressed is crucial as a benchmark for further studies. Unfortunately, membrane proteins are the category of proteins hardest to detect using conventional cellular proteomics protocols. In part, this is due to the hydrophobicity of membrane proteins as well as their general lower absolute abundance, which hinders detection. Here, we performed a combination of whole cell lysate proteomics and proteomics of membrane extracts solubilised with either SDS or FOS-choline-at various temperatures. The combined methods led to the detection of membrane proteins containing at least a single transmembrane helix (TMH). Within this data set we revealed a full oxidative phosphorylation pathway as well as an alternative NADH dehydrogenase type II (Ndh-) and a microaerophilic cytochrome oxidase ba. We also observed C. thermarum TA .A expressing transporters for ectoine and glycine betaine, compounds that are known osmolytes that may assist in maintaining a near neutral internal pH when the external pH is highly alkaline.
Applied and Environmental Microbiology, Oct 15, 2010
The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknow... more The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknown. We demonstrate that alkaliphilic (pH > 9) Bacillus species are sensitive to artificial iron (Fe 3؉) chelators and produce iron-chelating molecules. These alkaliphilic siderophores contain catechol and hydroxamate moieties, and their synthesis is stimulated by manganese(II) salts and suppressed by FeCl 3 addition. Purification and mass spectrometric characterization of the siderophore produced by Caldalkalibacillus thermarum failed to identify any matches to previously observed fragmentation spectra of known siderophores, suggesting a novel structure.
It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F 1 F o ATP syntha... more It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F 1 F o ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F 1 F o ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F 1 F o ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg 2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F 1 F o ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg 2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by secondshell residues thus the function of the ε subunit changes with growth conditions.
ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found i... more ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found in living cells. These enzymes are driven by an electrochemical ion gradient, which allows the catalytic evolution of ATP by a binding change mechanism. Most ATP synthases are capable of catalysing ATP hydrolysis to varying degrees, and to prevent wasteful ATP hydrolysis, bacteria and mitochondria have regulatory mechanisms such as ADP inhibition. Additionally, 1 subunit inhibition has also been described in three bacterial systems, Escherichia coli, Bacillus PS3 and Caldalkalibacillus thermarum TA2.A1. Previous studies suggest that the 1 subunit is capable of undergoing an ATP-dependent conformational change from the ATP hydrolytic inhibitory 'extended' conformation to the ATP-induced non-inhibitory 'hairpin' conformation. A recently published crystal structure of the F 1 domain of the C. thermarum TA2.A1 F 1 F o ATP synthase revealed a mutant 1 subunit lacking the ability to bind ATP in a hairpin conformation. This is a surprising observation considering it is an organism that performs no ATP hydrolysis in vivo, and appears to challenge the current dogma on the regulatory role of the 1 subunit. This has prompted a re-examination of present knowledge of the 1 subunits role in different organisms. Here, we compare published biochemical, biophysical and structural data involving 1 subunitmediated ATP hydrolysis regulation in a variety of organisms, concluding that the 1 subunit from the bacterial F-type ATP synthases is indeed capable of regulating ATP hydrolysis activity in a wide variety of bacteria, making it a potentially valuable drug target, but its exact role is still under debate.
Biochimica Et Biophysica Acta - Bioenergetics, Sep 1, 2018
a mutation in this gene (m.8969GNA), leading to replacement of a highly conserved serine residue ... more a mutation in this gene (m.8969GNA), leading to replacement of a highly conserved serine residue into asparagine at amino acid position 148, in a 14-year-old Chinese female who initially developed an isolated nephropathy followed by a complex clinical presentation with brain and muscle problems [1]. An equivalent of this mutation in yeast (atp6-S175N) was shown to prevent F O-mediated proton transfer and weaken F O to F 1 association. Herein we identified four first-site intragenic suppressors (aN 175 D, aN 175 K, aN 175 I, and aN 175 T), which, in light of a recently published atomic structure of yeast F O , provides strong indication that the detrimental consequences of the original mutation result from the establishment of a hydrogen bound between aN 175 and a nearby glutamate residue (aE 172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS 175 N mutation can be suppressed by second-site suppressors (aP 12 S, aI 171 F, aI 171 N, aI 239 F, and aI 200 M), of which some are very distantly located (by 20-30 Å) from the original mutation. The possibility to disrupt a pathogenic hydrogen bond through long-range effects is an interesting observation that holds promise for the development of therapeutic molecules.
Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of... more Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent K M value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo 3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo 3. Cardiolipin (CL) is an anionic phospholipid that consists of two phosphatidyl groups connected by a glycerol moiety. CL is important for optimal function of various eukaryotic and prokaryotic membrane protein complexes 1-5. CL can interact with bacterial respiratory complexes from phylogenetically diverse species, such as Mycobacterium phlei 6 , Rhodobacter sphaeroides 7 and Escherichia coli 8. Among E. coli respiratory chain complexes, CL was shown to activate purified, detergent-free cytochrome bo 3 9 and was the most efficient phospholipid for activation of detergent-solubilized NADH dehydrogenase 10 and of liposome-reconstituted nitrate reductase 11. Defined binding sites for CL have been determined in crystal structures of E. coli formate dehydrogenase N 12 , succinate dehydrogenase 13 , and nitrate reductase 11. The respiratory chain in Escherichia coli features a heme-copper-type terminal oxidase, cytochrome bo 3 , which transfers electrons from quinol-type substrates onto molecular oxygen. Next to this energetically efficient terminal oxidase, E. coli utilizes cytochrome bd as an alternative branch of the respiratory chain. Cytochrome bd oxidizes quinols, like ubiquinol or menaquinol, coupled with reduction of molecular oxygen to water (Fig. 1A) 14,15. Cytochrome bd is particularly important under conditions of stress, such as O 2-limitation 16 , in the presence of nitric oxide 17,18 hydrogen peroxide 19-21 and hydrogen sulfide 22,23. Lack of cytochrome bd in uropathogenic E. coli strains led to attenuation in mouse infection models 24. Cytochrome bd is present in of a broad variety of Gram-positive and Gram-negative bacteria and archea, but not in the respiratory chain of eukaryotes 14. Purified active cytochrome bd has been prepared from several bacterial species, including E. coli 25,26 , Azotobacter vinelandii 27 , Corynebacterium glutamicum 28 and Geobacillus thermodenitrificans 29,30. However, to our knowledge there are no data available concerning the effect of CL on cytochrome bd activity. In this report, we investigated the influence of CL on detergent-purified cytochrome bd. We found that CL activated the enzymatic activity of cytochrome bd from E. coli, G. thermodenitrificans and C. glutamicum. We then extended our experimentation and also assessed the impact of CL on the activity of purified cytochrome bo 3 from E. coli.
Abstract The multiwall carbon nanotubes-polypyrrole (MWCNTs-PPy) composite is a well-known hybrid... more Abstract The multiwall carbon nanotubes-polypyrrole (MWCNTs-PPy) composite is a well-known hybrid material suitable for energy storage applications such as capacitors. Since the electrochemical activity, and long-term stability of carbon-PPy systems are critical for their application, we examined the effect of grafting PPy via a 3-ABA linker with MWCNTs with respect to the preference of the donor–acceptor pairs, and furthermore, the durability. X-ray photoelectron spectroscopy studies of C 1s and O 1s signals reveal the electronic interaction between carbon and polymer (π−π* stacking), as well as a binding energy shift for C 1s and O 1s carbonyl signals due to the chemical bonding via the electron donating linker and grafted polymer. Cyclic voltammetry and galvanostatic charge–discharge studies of the capacitor electrode reveal the effect of the reversal doping of the polymer with chloride, as well as the synergy of double layer capacitance (MWCNTs) and pseudo-capacitance of the poly(pyrrole). Impedance spectroscopy studies confirm the improved electrochemical stability for the composite (stable until 1.2 V) in comparison to the bare polymer (degradation at 0.58 V).
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The ε subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevaili... more The ε subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevailing evidence supports the notion that when the ATP concentration falls below a certain threshold, the ε subunit changes its conformation from a non-inhibitory down-state to an extended up-state which then inhibits enzymatic ATP hydrolysis by binding to the catalytic domain. It has been demonstrated that the ε subunit from Bacillus PS3 is selective for ATP over other nucleotides, including GTP. In this study, the purine triphosphate selectivity is rationalized via results from MD simulations and free energy calculations for the R103A/R115A mutant of the ε subunit from Bacillus PS3, which binds ATP stronger than the wild-type (WT) protein. Our results are in good agreement with experimental data, and the elucidated molecular basis for selectivity could help to guide the design of novel GTP sensors.
Proceedings of the National Academy of Sciences, 2020
Significance F-ATP synthase is a fundamental enzyme supplying adenosine triphosphate (ATP), sprea... more Significance F-ATP synthase is a fundamental enzyme supplying adenosine triphosphate (ATP), spreading across all kingdoms of life. Despite remarkable conservation of its basic structure and function, biophysical studies have revealed discrete differences in the rotary mechanisms of bacterial and eukaryotic F 1 -ATPases (the catalytic portions of the enzymes). Here, we analyzed the rotational dynamics of Paracoccus denitrificans F 1 (PdF 1 ), a bacterial F 1 -ATPase that exhibits high homology with the core functional subunits of its mitochondrial counterpart. Notably, PdF 1 possesses a simplified chemomechanical scheme different from that of all other F 1 -ATPases. Our results reveal an unexpected diversity in the chemomechanical coupling cycle of the F 1 -ATPase machinery and show that features such as homology or phylogenetic relationship cannot uniquely define the rotary scheme pattern.
Diastereomers are characterised by an intrinsic energy difference, and thermodynamics dictate the... more Diastereomers are characterised by an intrinsic energy difference, and thermodynamics dictate their distribution within a dynamic equilibrium. The characteristic mechanistic reversibility and non‐ideal stereoselectivity of catalysts therefore simultaneously promote both synthesis and epimerization of products during the formation of diastereomers. This feature can even result in the thermodynamic inversion of a chiral centre against the catalyst's stereoselectivity. Here, we provide a comprehensive experimental and theoretical study of factors that govern thermodynamic epimerization in catalysis, using enzymes as example. Our analysis highlights, that the deduction of a catalyst's stereoselectivity based on the absolute configuration of the isolated product constitutes a potential pitfall. The selective formation of either the thermodynamic‐, or the kinetic product is less determined by the catalyst, but rather by the reaction conditions. Next to low temperatures, a high max...
A novel large-scale survey approach for microbial nonulosonic acids (sialic acids) including a fi... more A novel large-scale survey approach for microbial nonulosonic acids (sialic acids) including a first molecular level comparative study is presented.
The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP),... more The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can trans...
The digestive enzyme papain can drive the formation of crystalline titanium dioxide nanoparticles... more The digestive enzyme papain can drive the formation of crystalline titanium dioxide nanoparticles on surfaces. This dual functionality of thin film formation and mineralization promotion has the potential to enable the construction of hierarchical inorganic/organic structures in the form of continuous amorphous titania/protein films.
The aerobic thermoalkaliphile Caldalkalibacillus thermarum strain TA2.A1 is a member of a separat... more The aerobic thermoalkaliphile Caldalkalibacillus thermarum strain TA2.A1 is a member of a separate order of alkaliphilic bacteria closely related to the Bacillales order. Efforts to relate the genomic information of this evolutionary ancient organism to environmental adaptation have been thwarted by the inability to construct a complete genome. The existing draft genome is highly fragmented due to repetitive regions, and gaps between and over repetitive regions were unbridgeable. To address this, Oxford Nanopore Technology’s MinION allowed us to span these repeats through long reads, with over 6000-fold coverage. This resulted in a single 3.34 Mb circular chromosome. The profile of transporters and central metabolism gives insight into why the organism prefers glutamate over sucrose as carbon source. We propose that the deamination of glutamate allows alkalization of the immediate environment, an excellent example of how an extremophile modulates environmental conditions to suit its...
The class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hyd... more The class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hydroxypyruvate as nucleophilic donor substrate, giving access to synthetically challenging 3,4dihydroxy-α-ketoacids. The crystal structure of holo-SwHKA in complex with hydroxypyruvate revealed CH-π interactions between the CÀ H bonds at C3 of hydroxypyruvate and a phenylalanine residue at position 210, which in this case occupies the position of a conserved leucine residue. Mutagenesis to tyrosine further increased the electron density of the interacting aromatic system and effected a rate enhancement by twofold. While the leucine variant efficiently catalyses the enolisation of hydroxypyruvate as the first step in the aldol reaction, the enol intermediate then becomes trapped in a disfavoured configuration that considerably hinders subsequent CÀ C bond formation. In SwHKA, micromolar concentrations of inorganic phosphate increase the catalytic rate constant of enolisation by two orders of magnitude. This rate enhancement was now shown to be functionally conserved across the structurally distinct (α/β) 8 barrel and αββα sandwich folds of two pyruvate aldolases. Characterisation of the manganese (II) cofactor by electron paramagnetic resonance excluded ionic interactions between the metal centre and phosphate. Instead, histidine 44 was shown to be primarily responsible for the binding of phosphate in the micromolar range and the observed rate enhancement in SwHKA.
Materials for Renewable and Sustainable Energy, 2015
Nitrogen-doped carbon is a promising metalfree catalyst for oxygen reduction reaction in fuel cel... more Nitrogen-doped carbon is a promising metalfree catalyst for oxygen reduction reaction in fuel cells and metal-air batteries. However, its practical application necessitates a significant cost reduction, which can be achieved in part by using new synthetic methods and improvement of catalytic activity by increasing the density of redox active centers. This can be modulated by using polymer as the carbon and nitrogen sources. Although, superior catalytic activity of such N-doped C has been investigated in details, the electrochemical long-term stability of polymer-derived doped-carbon is still unclear. Herein, in this study we generated N-doped carbon from the most recommended polymer that is comparable to the state-of-the-art materials with porosity as high as 2,086 m 2 g-1 and a nitrogen doping level of 3-4 at.%, of which 56 % is pyrrolic N, 36.1 % pyridinic and *8 % graphitic. The electrochemical characterization shows that N-doped carbon is catalytic toward oxygen reduction in an alkaline electrolyte via a favorable four-electron process, however, not stable under long-term potential scanning. The irreversible electrochemical oxidation of this material is associated with the presence of a significant content or pyrrolic and pyridinic N close to the edge of the carbon network originating from the polypyrrole precursor. These structures are less stable under operating electrochemical potential. The role of polypyrrole as the precursor of N-doped carbons has to be carefully revised since it supplies sufficient number of catalytic sites, but also generates unstable functionalities on the carbon surface.
Protein-protein interactions are well-known to regulate enzyme activity in cell signaling and met... more Protein-protein interactions are well-known to regulate enzyme activity in cell signaling and metabolism. Here, we show that protein-protein interactions regulate the activity of a respiratory-chain enzyme, CymA, by changing the direction or bias of catalysis. CymA, a member of the widespread NapC/NirT superfamily, is a menaquinol-7 (MQ-7) dehydrogenase that donates electrons to several distinct terminal reductases in the versatile respiratory network of Shewanella oneidensis.W e report the incorporation of CymA within solid-supported membranes that mimic the inner membrane architecture of S. oneidensis. Quartz-crystal microbalance with dissipation (QCM-D) resolved the formation of a stable complex between CymA and one of its native redox partners, flavocytochrome c 3 (Fcc 3 ) fumarate reductase. Cyclic voltammetry revealed that CymA alone could only reduce MQ-7, while the CymA-Fcc 3 complex catalyzed the reaction required to support anaerobic respiration, the oxidation of MQ-7. We propose that MQ-7 oxidation in CymA is limited by electron transfer to the hemes and that complex formation with Fcc 3 facilitates the electron-transfer rate along the heme redox chain. These results reveal a yet unexplored mechanism by which bacteria can regulate multibranched respiratory networks through protein-protein interactions.
Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the mol... more Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged Cterminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.
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Papers by Duncan McMillan