A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency.

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

Structural and Functional Characterization of NadR from Lactococcus lactis.

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a 'sink' for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains.

The twisted relation between Pnu and SWEET transporters.

The evolutionary relation between sugar and vitamin transporters from the SWEET and Pnu families is unclear. They have similar 3D structures, but differ in the topology of their secondary structure elements, and lack significant sequence similarity. Here we analyze the structures and sequences of different members of the SWEET and Pnu transporter families and propose an evolutionary pathway by which they may have diverged from a common ancestor. A 3D domain swapping event can explain the topological differences between the families, as well as the puzzling observation that a highly conserved and essential sequence motif of the SWEET family (the PQ loop) is absent from the Pnu transporters.

Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters.

Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter GltPh. Each subunit in the homotrimeric GltPh supports the coupled translocation of one aspartate molecule and three Na(+) ions as well as an uncoupled flux of Cl(-) ions. Recent crystal structures of GltPh revealed three possible conformations for the subunits, but it is unclear whether the motions of individual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in individual GltPh trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three GltPh subunits undergo conformational changes stochastically and independently of each other.

An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates.

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria.

Targeted Diazotransfer Reagents Enable Selective Modification of Proteins with Azides.

In chemical biology, azides are used to chemically manipulate target structures in a bioorthogonal manner for a plethora of applications ranging from target identification to the synthesis of homogeneously modified protein conjugates. While a variety of methods have been established to introduce the azido group into recombinant proteins, a method that directly converts specific amino groups in endogenous proteins is lacking. Here, we report the first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies the model proteins streptavidin and avidin and the membrane protein BioY on cell surface. The reagent converts amines in the proximity of the binding pocket to azides and leaves the remaining amino groups in streptavidin untouched. Reagents of this novel class will find use in target identification as well as the selective functionalization and bioorthogonal protection of proteins.

The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli.

BACKGROUND: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector. RESULTS: By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein. CONCLUSIONS: Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).

Relative Rates of Amino Acid Import via the ABC Transporter GlnPQ Determine the Growth Performance of Lactococcus lactis.

UNLABELLED: The GlnPQ transporter from Lactococcus lactis has the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate. IMPORTANCE: GlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, including L. lactis and several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine.

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

GltPh from Pyrococcus horikoshii is a homotrimeric Na(+)-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na(+) and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na(+) binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m(-1)s(-1)). At least two sodium ions bind before aspartate. The binding of Na(+) requires a very high activation energy (Ea 106.8 kJ mol(-1)) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na(+) concentration present. Binding of aspartate was not observed in the absence of Na(+), whereas in the presence of high Na(+) concentrations (above the Kd for Na(+)) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 10(5) m(-1)s(-1)), with low Ea and Q10 values (42.6 kJ mol(-1) and 1.8, respectively). We conclude that Na(+) binding is most likely the rate-limiting step for substrate binding.

Diversity of membrane transport proteins for vitamins in bacteria and archaea.

BACKGROUND: All organisms use cofactors to extend the catalytic capacities of proteins. Many bacteria and archaea can synthesize cofactors from primary metabolites, but there are also prokaryotes that do not have the complete biosynthetic pathways for all essential cofactors. These organisms are dependent on the uptake of cofactors, or at least their precursors that cannot be synthesized, from the environment. Even in those organisms that contain complete biosynthetic pathways membrane transporters are usually present, because the synthesis of cofactors is more costly than uptake. SCOPE OF REVIEW: Here we give an overview of bacterial and archaeal transport systems for B-type vitamins, which are either cofactors or precursors thereof. MAJOR CONCLUSIONS: Prokaryotic vitamin transporters are extremely diverse, and found in many families of transporters. A few of these transport systems have been characterized in detail, but for most of them mechanistic insight is lacking. GENERAL SIGNIFICANCE: The lack of structural and functional understanding of bacterial vitamin transporters is unfortunate because they may be targets for new antibiotics. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins. Guest Editor: Bjorn Pedersen.

Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters.

Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications.

High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG.

BACKGROUND: For membrane protein production, the Escherichia coli T7 RNA polymerase (T7 RNAP)-based protein production strain BL21(DE3) in combination with T7-promoter based expression vectors is widely used. Cells are routinely cultured in Lysogeny broth (LB medium) and expression of the chromosomally localized t7rnap gene is governed by the isopropyl-ß-D-1-thiogalactopyranoside (IPTG) inducible lacUV5 promoter. The T7 RNAP drives the expression of the plasmid borne gene encoding the recombinant membrane protein. Production of membrane proteins in the cytoplasmic membrane rather than in inclusion bodies in a misfolded state is usually preferred, but often hampered due to saturation of the capacity of the Sec-translocon, resulting in low yields. RESULTS: Contrary to expectation we observed that omission of IPTG from BL21(DE3) cells cultured in LB medium can lead to significantly higher membrane protein production yields than when IPTG is added. In the complete absence of IPTG cultures stably produce membrane proteins in the cytoplasmic membrane, whereas upon the addition of IPTG membrane proteins aggregate in the cytoplasm and non-producing clones are selected for. Furthermore, in the absence of IPTG, membrane proteins are produced at a lower rate than in the presence of IPTG. These observations indicate that in the absence of IPTG the Sec-translocon capacity is not/hardly saturated, leading to enhanced membrane protein production yields in the cytoplasmic membrane. Importantly, for more than half of the targets tested the yields obtained using un-induced BL21(DE3) cells were higher than the yields obtained in the widely used membrane protein production strains C41(DE3) and C43(DE3). Since most secretory proteins reach the periplasm via the Sec-translocon, we also monitored the production of three secretory recombinant proteins in the periplasm of BL21(DE3) cells in the presence and absence of IPTG. For all three targets tested omitting IPTG led to the highest production levels in the periplasm. CONCLUSIONS: Omission of IPTG from BL21(DE3) cells cultured in LB medium provides a very cost- and time effective alternative for the production of membrane and secretory proteins. Therefore, we recommend that this condition is incorporated in membrane- and secretory protein production screens.

Structural divergence of paralogous S components from ECF-type ABC transporters.

Energy coupling factor (ECF) proteins are ATP-binding cassette transporters involved in the import of micronutrients in prokaryotes. They consist of two nucleotide-binding subunits and the integral membrane subunit EcfT, which together form the ECF module and a second integral membrane subunit that captures the substrate (the S component). Different S components, unrelated in sequence and specific for different ligands, can interact with the same ECF module. Here, we present a high-resolution crystal structure at 2.1 Å of the biotin-specific S component BioY from Lactococcus lactis. BioY shares only 16% sequence identity with the thiamin-specific S component ThiT from the same organism, of which we recently solved a crystal structure. Consistent with the lack of sequence similarity, BioY and ThiT display large structural differences (rmsd = 5.1 Å), but the divergence is not equally distributed over the molecules: The S components contain a structurally conserved N-terminal domain that is involved in the interaction with the ECF module and a highly divergent C-terminal domain that binds the substrate. The domain structure explains how the S components with large overall structural differences can interact with the same ECF module while at the same time specifically bind very different substrates with subnanomolar affinity. Solitary BioY (in the absence of the ECF module) is monomeric in detergent solution and binds D-biotin with a high affinity but does not transport the substrate across the membrane.

Structural basis for arabinoxylo-oligosaccharide capture by the probiotic Bifidobacterium animalis subsp. lactis Bl-04.

Glycan utilization plays a key role in modulating the composition of the gut microbiota, but molecular insight into oligosaccharide uptake by this microbial community is lacking. Arabinoxylo-oligosaccharides (AXOS) are abundant in the diet, and are selectively fermented by probiotic bifidobacteria in the colon. Here we show how selectivity for AXOS uptake is established by the probiotic strain Bifidobacterium animalis subsp. lactis Bl-04. The binding protein BlAXBP, which is associated with an ATP-binding cassette (ABC) transporter that mediates the uptake of AXOS, displays an exceptionally broad specificity for arabinosyl-decorated and undecorated xylo-oligosaccharides, with preference for tri- and tetra-saccharides. Crystal structures of BlAXBP in complex with four different ligands revealed the basis for this versatility. Uniquely, the protein was able to recognize oligosaccharides in two opposite orientations, which facilitates the optimization of interactions with the various ligands. Broad substrate specificity was further enhanced by a spacious binding pocket accommodating decorations at different mainchain positions and conformational flexibility of a lid-like loop. Phylogenetic and genetic analyses show that BlAXBP is highly conserved within Bifidobacterium, but is lacking in other gut microbiota members. These data indicate niche adaptation within Bifidobacterium and highlight the metabolic syntrophy (cross-feeding) among the gut microbiota.

The S-component fold: a link between bacterial transporters and receptors.

The processes of nutrient uptake and signal sensing are crucial for microbial survival and adaptation. Membrane-embedded proteins involved in these functions (transporters and receptors) are commonly regarded as unrelated in terms of sequence, structure, mechanism of action and evolutionary history. Here, we analyze the protein structural universe using recently developed artificial intelligence-based structure prediction tools, and find an unexpected link between prominent groups of microbial transporters and receptors. The so-called S-components of Energy-Coupling Factor (ECF) transporters, and the membrane domains of sensor histidine kinases of the 5TMR cluster share a structural fold. The discovery of their relatedness manifests a widespread case of prokaryotic "transceptors" (related proteins with transport or receptor function), showcases how artificial intelligence-based structure predictions reveal unchartered evolutionary connections between proteins, and provides new avenues for engineering transport and signaling functions in bacteria.

Structure and mode of peptide binding of pheromone receptor PrgZ.

We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.

The structural basis for peptide selection by the transport receptor OppA.

Oligopeptide-binding protein A (OppA) from Lactococcus lactis binds peptides of an exceptionally wide range of lengths (4-35 residues), with no apparent sequence preference. Here, we present the crystal structures of OppA in the open- and closed-liganded conformations. The structures directly explain the protein's phenomenal promiscuity. A huge cavity allows binding of very long peptides, and a lack of constraints for the position of the N and C termini of the ligand is compatible with binding of peptides with varying lengths. Unexpectedly, the peptide's amino-acid composition (but not the exact sequence) appears to have a function in selection, with a preference for proline-rich peptides containing at least one isoleucine. These properties can be related to the physiology of the organism: L. lactis is auxotrophic for branched chain amino acids and favours proline-rich caseins as a source of amino acids. We propose a new mechanism for peptide selection based on amino-acid composition rather than sequence.

Physiological adaptation of the bacterium Lactococcus lactis in response to the production of human CFTR.

Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that different strategies are needed to overcome low expression yields and toxicity.

A Hitchhiker's Guide to Supplying Enzymatic Reducing Power into Synthetic Cells.

The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-of-equilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P)+. In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P)+ coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes─transhydrogenases─to overcome this potential bottleneck.