ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism

Karpowich, Nathan K.; Song, Jin Mei; Cocco, Nicolette; Wang, Da-Neng

doi:10.1038/nsmb.3040

Cited by 38 publications

(84 citation statements)

References 50 publications

(107 reference statements)

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“…In a recent paper, the structure of the riboflavin-binding subunit Lmo1945 (EcfS or RibU) of the riboflavin uptake system was reported (25). Physiological experiments showing that Lmo1945 is indeed involved in riboflavin transport were not performed (25), and we set out to validate this anticipated function.…”

Section: Resultsmentioning

confidence: 99%

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

et al. 2016

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The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus. Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B 2 ), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. R iboflavin (vitamin B 2 ) is not synthesized by mammals but is synthesized by many microorganisms and by all plants (1).The genomes of the human bacterial pathogens Listeria monocytogenes, Streptococcus pyogenes, and Enterococcus faecalis do not contain genes encoding riboflavin biosynthetic enzymes (2), and thus these microorganisms are riboflavin auxotrophs. L. monocytogenes, Streptococcus pyogenes, and Enterococcus faecalis produce energy-coupling factor (ECF) transporters which combine with a riboflavin-specific binding subunit (subunit EcfS or RibU) (3), and ECF-RibU-mediated uptake is the sole source of riboflavin (4).Riboflavin cannot be detected in cell extracts of bacteria (5-7), indicating that it is completely metabolized by flavokinases (RibF) (EC 2.7.1.26) and FAD synthetases (RibC) (EC 2.7.7.2). These enzymes catalyze the formation of FMN (from riboflavin and ATP) and FAD (from FMN and ATP) (see Fig. S1 in the supplemental material) and play an important role in metabolic trapping of riboflavin (8). In bacteria, bifunctional flavokinases/FAD synthetases have been found, whereas in eukarya and archaea, monofunctional enzymes seem to be the rule (9). FMN and FAD are cofactors of flavoproteins/flavoenzymes, which have a wide variety of different biological functions (10). The number of flavindependent proteins varies greatly in different organisms (and among pathogens) and covers a range from approximately 0.1% to 3.5% of the proteome (11). In Escherichia coli, FMN and FAD are present at levels which are about 30 times lower than those of the highly abundant amino acids or nucleotides (12), whereby FAD (170 M) clearly is present at higher levels than FMN (54

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Section: Resultsmentioning

confidence: 99%

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

et al. 2016

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“…We took advantage of the observations that while isolated S subunits co-purify with their respective transport substrate [10–12, 15, 21, 37] complete ECF transporters do not [15–18, 25, 37, 38]. We previously demonstrated that the homologous ECF riboflavin transporter from Listeria monocytogenes , LmECF-RibU, captures the riboflavin transport substrate by a “release and catch” mechanism [37]. In this system, ATP binding drives a conformational change that dissociates the EcfS subunit from the EcfAA′T ECF module.…”

Section: Resultsmentioning

confidence: 99%

“…Several studies indicated that S subunits for different vitamins compete for transport by the ECF module and such competition is enhanced in the presence of the substrate [9, 37, 55]. These observations suggest that the apo and substrate-bound conformations of EcfS have different affinities for the ECF module.…”

Section: Discussionmentioning

confidence: 99%

“…The LmECF-RibU transporter with His199Ala and His206Ala of EcfA′ and EcfA, respectively, was incubated with 2mM MgATP and various concentrations of riboflavin, as described in [37]. Subsequently, 50 μl of each sample was injected on a Shodex KW804 analytical SEC column (Thomson, Clear Brook, VA) equilibrated with 50 mM Tris, pH 7.5, 200 mM Na 2 SO 4 , 3 mM NaN 3 and 0.05 % DDM on an HPLC (Waters, Milford, MA) equipped with UV and fluorescence detectors.…”

Section: Methodsmentioning

confidence: 99%

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An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

Karpowich

Song

Wang

2016

Journal of Molecular Biology

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ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA′). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from T. maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein-substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound occluded state, and nonconservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

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“…An important question that still needs to be addressed is in which conformational state the ECF module interacts with the S‐component. It needs to be demonstrated whether the toppling of the S‐component might depend on the hydrolysis of ATP as it has been postulated by alternative models …”

Section: Energy‐coupling Factor and Adenosine 5′‐triphosphate‐bindingmentioning

confidence: 99%

Energy‐Coupling Factor Transporters as Novel Antimicrobial Targets

Bousis

Setyawati

Diamanti

et al. 2018

Advanced Therapeutics

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In an attempt to find new antibiotics, novel ways of interfering with important biological functions should be explored, especially with those which are necessary or even irreplaceable for bacterial survival, growth, and virulence. The purpose of this review is to highlight B-type vitamin transporters from the energy-coupling factor (ECF) family, which are not present in humans, as potential antimicrobial targets. In addition, a druggability analysis of an ECF transporter for folic acid and sequence-conservation studies in seven prominent pathogens revealed new druggable pockets. Evaluation of the presence of de novo biosynthetic routes for the vitamins in question in the seven pathogens confirmed that this target class holds promise for the discovery of antimicrobial drugs with a new mechanism of action, possibly on a broad-spectrum level.

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ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism

Cited by 38 publications

References 50 publications

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

Energy‐Coupling Factor Transporters as Novel Antimicrobial Targets

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