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Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared. [ABSTRACT FROM AUTHOR]

Membrane‐bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H+ pump that transports the H+ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na+ via a hypothetical "billiard‐type" mechanism, also involving the hydrolysis‐generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid‐quench and pulse‐chase measurements with [32P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate‐limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme‐substrate complex. Na+, whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H2O with D2O decreased the rates of pyrophosphate hydrolysis by both Na+‐ and H+‐transporting bacterial mPPases, the effect being more significant than with a non‐transporting soluble pyrophosphatase. We also show that the Na+‐pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis‐generated proton both in H+‐pumping and Na+‐pumping by mPPases. [ABSTRACT FROM AUTHOR]

Membrane pyrophosphatases (mPPases) found in plant vacuoles and some prokaryotes and protists are ancient cation pumps that couple pyrophosphate hydrolysis with the H+ and/or Na+ transport out of the cytoplasm. Because this function is reversible, mPPases play a role in maintaining the level of cytoplasmic pyrophosphate, a known regulator of numerous metabolic reactions. mPPases arouse interest because they are among the simplest membrane transporters and have no homologs among known ion pumps. Detailed phylogenetic studies have revealed various subtypes of mPPases and suggested their roles in the evolution of the "sodium" and "proton" bioenergetics. This treatise focuses on the mechanistic aspects of the transport reaction, namely, the coupling step, the role of the chemically produced proton, subunit cooperation, and the relationship between the proton and sodium ion transport. The available data identify H+-PPases as the first non-oxidoreductase pump with a "direct-coupling" mechanism, i.e., the transported proton is produced in the coupled chemical reaction. They also support a "billiard" hypothesis, which unifies the H+ and Na+ transport mechanisms in mPPase and, probably, other transporters. [ABSTRACT FROM AUTHOR]

Two newly isolated bacterial membrane-bound pyrophosphatase from the mesophile Methanosarcina mazei and the moderate thermophile Moorella thermoacetica that catalyze [Na.sup.} rather than [H.sup.} transport into Escherichia coli inner membrane vesicles (IMV) is demonstrated.

The dependency of the ligand Methanosarcina mazei-pyrophosphatase (Mm-PPase) [PP.sub.i]-hydrolyzing function and protection offered against Mm-PPase inactivation by trypsin and the SH-reagent mersalyl is analyzed to understand the relationships among [Na.sup.+], [K.sup.+], [Mg.sup.2+], and [PP.sub.i] binding sites. The data obtained provide insight into the considerable flexibility of Mm-PPase structure and its cyclic change in the course of catalytic turnover.

An expression of the hyperthermophilic bacterium, Thermotoga maritima (Tm-PPase) in Escherichia coli results in a higher yield than in yeast, allowing a detailed characterization of the enzyme. It is also shown that Tm-PPase has an absolute requirement for Na(super +) and displays maximal activity in the presence of millimolar levels of both Na(super +) and K(super +).

Membrane-bound pyrophosphatases (mPPases) hydrolyze pyrophosphate (PPi) to transport H+, Na+ or both and help organisms to cope with stress conditions, such as high salinity or limiting nutrients. Recent elucidation of mPPase structure and identification of subfamilies that have fully or partially switched from Na+ to H+ pumping have established mPPases as versatile models for studying the principles governing the mechanism, specificity and evolution of cation transporters. In the present study, we constructed an accurate phylogenetic map of the interface of Na+-transporting PPases (Na+-PPases) and Na+- and H+-transporting PPases (Na+,H+-PPases), which guided our experimental exploration of the variations in PPi hydrolysis and ion transport activities during evolution. Surprisingly, we identified two mPPase lineages that independently acquired physiologically significant Na+ and H+ cotransport function. Na+,H+-PPases of the first lineage transport H+ over an extended [Na+] range, but progressively lose H+ transport efficiency at high [Na+]. In contrast, H+-transport by Na+,H+-PPases of the second lineage is not inhibited by up to 100 mM Na+. With the identification of Na+,H+-PPase subtypes, the mPPases protein superfamily appears as a continuum, ranging from monospecific Na+ transporters to transporters with tunable levels of Na+ and H+ cotransport and further to monospecific H+ transporters. Our results lend credence to the concept that Na+ and H+ are transported by similar mechanisms, allowing the relative efficiencies of Na+ and H+ transport to be modulated by minor changes in protein structure during the course of adaptation to a changing environment. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ionpumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme. [ABSTRACT FROM AUTHOR]

Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+ -transporting PPases are divided into phylogenetically distinct K+ -independent and K+ - dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%-34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in 'canonical' mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+ - dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+ -PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2-PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+ -transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+ - transporting mPPases. [ABSTRACT FROM AUTHOR]

In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H+ transport across biological membranes (H+-pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na+ (Na+-pyrophosphatase) or both Na+ and H+ (Na+,H+-pyrophosphatase). Both these transporters require Na+ for pyrophosphate hydrolysis and are further activated by K+. The determination of the three-dimensional structures of H+- and Na+-pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms. [ABSTRACT FROM AUTHOR]

One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H+-transporting (found in prokaryotes, protists, and plants) and Na+-transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport. However, experiments using a fluorescent pH probe and 22Na+ measurements in the current study revealed that five bacterial PPases expressed in Escherichia coli have the ability to simultaneously translocate H+ and Na+ into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na+,H+-PPase subfamily, which apparently evolved from Na+-PPases. Interestingly, genes for Na+,H+-PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na+,H+-PPases require Na+ for hydrolytic and transport activities and are further activated by K+. Based on ionophore effects, we conclude that the Na+ and H+ transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na+,H+-PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na+ and H+ can be transported via the same mechanism, with switching between Na+ and H+ specificities requiring only subtle changes in the transporter structure. [ABSTRACT FROM AUTHOR]

Membrane-bound pyrophosphatase (PPase) is commonly believed to couple pyrophosphate (PPi) hydrolysis to H+ transport across the membrane. Here, we demonstrate that two newly isolated bacterial membrane PPases from the mesophile Methanosarcina mazei (Mm-PPase) and the moderate thermophile Moorella thermoacetica and a previously described PPase from the hyperthermophilic bacterium Thermotoga maritima catalyze Na+ rather than H+ transport into Escherichia coli inner membrane vesicles (IMV). When assayed in uncoupled IMV, the three PPases exhibit an absolute requirement for Na+ but display the highest hydrolyzing activity in the presence of both Na+ and K+. Steady-state kinetic analysis of PPi hydrolysis by Mm-PPase revealed two Na+ binding sites. One of these sites can also bind K+, resulting in a 10-fold increase in the affinity of the other site for Na+ and a 2-fold increase in maximal velocity. PPi-driven 22Na+ transport into IMV containing Mm-PPase was unaffected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, inhibited by the Na+ ionophore monensin, and activated by the K+ ionophore valinomycin. The Na+ transport was accompanied by the generation of a positive inside membrane potential as reported by Oxonol VI. These findings define Na++dependent PPases as electrogenic Na+ pumps. Phylogenetic analysis suggests that ancient gene duplication preceded the split of Na+ and H+-PPases. [ABSTRACT FROM AUTHOR]

H+-pyrophosphatase (H+-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H+-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu197, Glu202, Glu550, and Glu649 in the H+-PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PPi hydrolysis and PPi-energized proton translocation, indicating that the negative charge of Glu202 is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg2+ concentrations but were only slightly affected at high Mg2+ concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways. Substitution of Glu197, Glu202, and Glu649 resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methyl- enediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu550 and Glu649, R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PPi hydrolysis to proton transport. [ABSTRACT FROM AUTHOR]