Quantitative proteomics reveals oxygen-induced adaptations in TA2.A1 microaerobic chemostat cultures.

The thermoalkaliphile possesses a highly branched respiratory chain. These primarily facilitate growth at a wide range of dissolved oxygen levels. The aim of this study was to investigate the regulation of respiratory chain.

Membrane proteome of the thermoalkaliphile TA2.A1.

Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms. The thermoalkaliphile TA2.A1 is an organism of interest for studies into how alkaliphiles adapt to their extreme lifestyles, as it can grow from pH 7.

Rapid and Highly Stable Membrane Reconstitution by LAiR Enables the Study of Physiological Integral Membrane Protein Functions.

Functional reintegration into lipid environments represents a major challenge for investigation of integral membrane proteins (IMPs). Here, we report a new approach, termed LMNG Auto-insertion Reintegration (LAiR), for reintegration of IMPs into lipid bilayers within minutes. The resulting proteoliposomes displayed an unprecedented capability to maintain proton gradients and long-term stability.

ATP binding by an FF ATP synthase ε subunit is pH dependent, suggesting a diversity of ε subunit functional regulation in bacteria.

It is a conjecture that the ε subunit regulates ATP hydrolytic function of the FF 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.

Substrate Induced Movement of the Metal Cofactor between Active and Resting State.

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements.

The Profound Influence of Lipid Composition on the Catalysis of the Drug Target NADH Type II Oxidoreductase.

Lipids play a pivotal role in cellular respiration, providing the natural environment in which an oxidoreductase interacts with the quinone pool. To date, it is generally accepted that negatively charged lipids play a major role in the activity of quinone oxidoreductases. By changing lipid compositions when assaying a type II NADH:quinone oxidoreductase, we demonstrate that phosphatidylethanolamine has an essential role in substrate binding and catalysis.

Peripheral Membrane Proteins: Promising Therapeutic Targets across Domains of Life.

Membrane proteins can be classified into two main categories-integral and peripheral membrane proteins-depending on the nature of their membrane interaction. Peripheral membrane proteins are highly unique amphipathic proteins that interact with the membrane indirectly, using electrostatic or hydrophobic interactions, or directly, using hydrophobic tails or GPI-anchors. The nature of this interaction not only influences the location of the protein in the cell, but also the function.

Cardiolipin enhances the enzymatic activity of cytochrome bd and cytochrome bo solubilized in dodecyl-maltoside.

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.

Assessing the Thiamine Diphosphate Dependent Pyruvate Dehydrogenase E1 Subunit for Carboligation Reactions with Aliphatic Ketoacids.

The synthetic properties of the Thiamine diphosphate (ThDP)-dependent pyruvate dehydrogenase E1 subunit from (PDH E1) was assessed for carboligation reactions with aliphatic ketoacids. Due to its role in metabolism, PDH E1 was previously characterised with respect to its biochemical properties, but it was never applied for synthetic purposes. Here, we show that PDH E1 is a promising biocatalyst for the production of chiral α-hydroxyketones.

The 3 × 120° rotary mechanism of F-ATPase is different from that of the bacterial and mitochondrial F-ATPases.

The rotation of F-ATPase (PdF) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or below , PdF showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF executes binding, hydrolysis, and probably product release at the same dwell.

A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP.

Background: Prediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding.

Genomic analysis of Caldalkalibacillus thermarum TA2.A1 reveals aerobic alkaliphilic metabolism and evolutionary hallmarks linking alkaliphilic bacteria and plant life.

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 Molecular Basis for Purine Binding Selectivity in the Bacterial ATP Synthase ϵ Subunit.

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 that 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.

Tackling the chemical diversity of microbial nonulosonic acids - a universal large-scale survey approach.

Nonulosonic acids, commonly referred to as sialic acids, are a highly important group of nine-carbon sugars common to all domains of life. They all share biosynthetic and structural features, but otherwise display a remarkable chemical diversity. In humans, sialic acids cover all cells which makes them important for processes such as cellular protection, immunity and brain development.

Revealing the Membrane-Bound Catalytic Oxidation of NADH by the Drug Target Type-II NADH Dehydrogenase.

Type-II NADH:quinone oxidoreductases (NDH-2s) are an important element of microbial pathogen electron transport chains and an attractive drug target. Despite being widely studied, its mechanism and catalysis are still poorly understood in a hydrophobic membrane environment. A recent report for the NDH-2 showed NADH oxidation in a solution-based assay but apparently showed the reverse reaction in electrochemical studies, calling into question the validity of the electrochemical approach.

Ionophoric effects of the antitubercular drug bedaquiline.

Bedaquiline (BDQ), an inhibitor of the mycobacterial FF-ATP synthase, has revolutionized the antitubercular drug discovery program by defining energy metabolism as a potent new target space. Several studies have recently suggested that BDQ ultimately causes mycobacterial cell death through a phenomenon known as uncoupling. The biochemical basis underlying this, in BDQ, is unresolved and may represent a new pathway to the development of effective therapeutics.

Insights into the regulatory function of the subunit from bacterial F-type ATP synthases: a comparison of structural, biochemical and biophysical data.

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.

Catalytic robustness and torque generation of the F-ATPase.

The F-ATPase is the catalytic portion of the FF ATP synthase and acts as a rotary molecular motor when it hydrolyzes ATP. Two decades have passed since the single-molecule rotation assay of F-ATPase was established. Although several fundamental issues remain elusive, basic properties of F-type ATPases as motor proteins have been well characterized, and a large part of the reaction scheme has been revealed by the combination of extensive structural, biochemical, biophysical, and theoretical studies.

Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation.

FF ATP synthases are bidirectional molecular motors that translocate protons across the cell membrane by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acid/alkaline). Unlike mesophilic ATP synthases, alkaliphilic enzymes have tightly regulated ATP hydrolysis activity, which can be relieved in the presence of lauryldimethylamine oxide.

Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase.

Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored.

Population Changes in a Community of Alkaliphilic Iron-Reducing Bacteria Due to Changes in the Electron Acceptor: Implications for Bioremediation at Alkaline Cr(VI)-Contaminated Sites.

A serial enrichment culture has been grown in an alkaline Fe(III)-citrate-containing medium from an initial inoculum from a soil layer beneath a chromium ore processing residue (COPR) disposal site where Cr(III) is accumulating from Cr(VI) containing leachate. This culture is dominated by two bacterial genera in the order Clostridiales, , and an unnamed subgroup. This paper investigates the growth characteristics of the culture when Cr(VI) is added to the growth medium and when aquifer sand is substituted for Fe(III)-citrate.

Regulatory conformational changes of the ε subunit in single FRET-labeled FF-ATP synthase.

Subunit ε is an intrinsic regulator of the bacterial FF-ATP synthase, the ubiquitous membrane-embedded enzyme that utilizes a proton motive force in most organisms to synthesize adenosine triphosphate (ATP). The C-terminal domain of ε can extend into the central cavity formed by the α and β subunits, as revealed by the recent X-ray structure of the F portion of the enzyme. This insertion blocks the rotation of the central γ subunit and, thereby, prevents wasteful ATP hydrolysis.

The Impact of Enzyme Orientation and Electrode Topology on the Catalytic Activity of Adsorbed Redox Enzymes.

It is well established that the structural details of electrodes and their interaction with adsorbed enzyme influences the interfacial electron transfer rate. However, for nanostructured electrodes, it is likely that the structure also impacts on substrate flux near the adsorbed enzymes and thus catalytic activity. Furthermore, for enzymes converting macro-molecular substrates it is possible that the enzyme orientation determines the nature of interactions between the adsorbed enzyme and substrate and therefore catalytic rates.

Extracellular electron transport-mediated Fe(III) reduction by a community of alkaliphilic bacteria that use flavins as electron shuttles.

The biochemical and molecular mechanisms used by alkaliphilic bacterial communities to reduce metals in the environment are currently unknown. We demonstrate that an alkaliphilic (pH > 9) consortium dominated by Tissierella, Clostridium, and Alkaliphilus spp. is capable of using iron (Fe(3+)) as a final electron acceptor under anaerobic conditions.