24 research outputs found

    The Aspartate-Less Receiver (ALR) Domains: Distribution, Structure and Function

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    <div><p>Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from <i>Streptococcus pneumoniae</i> presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.</p></div

    ALR statistics and phylogeny.

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    <p>(<b>a</b>) Frequency of amino acid substitutions within six key ‘invariant’ REC residues in ALR sequences: the (now changed in ALRs) conserved histidine kinase phosphorylated aspartate residue position that defines the ALR subfamily (Phospho-Asp), acidic triad residue-1 (Glu9 in RitR) and acid triad residue-2 (Lys10 in RitR) that normally help coordinate the metal ion active pocket, the Tyrosine/Phenylalanine (Tyr/Phe, Tyr100 in RitR) and Threonine/Serine (Thr/Ser, Asp81 in RitR) that make up the Y/T-coupling system, and the conserved pocket Lys (Lys103 in RitR). Notice that where catalytic active pocket Asp/Lys residues have often been changed in ALR sequences (top panel), the T/Y-coupling residues generally remain conserved (bottom panel). This trend in conservation is also observed for the acidic triad-1 and the universally conserved pocket Lys residue (Lys103 in RitR), but not for acidic triad-2. (<b>b</b>) Taxonomic distribution of ALR sequences. The number of ALRs discovered in the given class or phylum is shown in parentheses. (<b>c</b>) Distribution of the average number of ALR sequences per completed genome by phyla. (<b>d</b>) Bar graph of the percentage contribution of a given Effector Domain (ED) within total REC sequences (shown as black bars) and ALR sequences only (shown as non-black bars). An asterisk above the bars indicates that ALRs are enriched for the ED by over 50% within the ALR population compared to their representation within typical REC sequence populations. An asterisk in front of the ED name indicates that the ALR or REC domain is (unusually) C-terminal to the ED sequence.</p

    <i>Streptomyces wadayamensis</i> MppP Is a Pyridoxal 5′-Phosphate-Dependent l‑Arginine α‑Deaminase, γ‑Hydroxylase in the Enduracididine Biosynthetic Pathway

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    l-Enduracididine (l-End) is a nonproteinogenic amino acid found in a number of bioactive peptides, including the antibiotics teixobactin, enduracidin, and mannopeptimycin. The potent activity of these compounds against antibiotic-resistant pathogens like MRSA and their novel mode of action have garnered considerable interest for the development of these peptides into clinically relevant antibiotics. This goal has been hampered, at least in part, by the fact that l-End is difficult to synthesize and not currently commercially available. We have begun to elucidate the biosynthetic pathway of this unusual building block. In mannopeptimycin-producing strains, like <i>Streptomyces wadayamensis</i>, l-End is produced from l-Arg by the action of three enzymes: MppP, MppQ, and MppR. Herein, we report the structural and functional characterization of MppP. This pyridoxal 5′-phosphate (PLP)-dependent enzyme was predicted to be a fold type I aminotransferase on the basis of sequence analysis. We show that MppP is actually the first example of a PLP-dependent hydroxylase that catalyzes a reaction of l-Arg with dioxygen to yield a mixture of 2-oxo-4-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid in a 1.7:1 ratio. The structure of MppP with PLP bound to the catalytic lysine residue (Lys221) shows that, while the tertiary structure is very similar to those of the well-studied aminotransferases, there are differences in the arrangement of active site residues around the cofactor that likely account for the unusual activity of this enzyme. The structure of MppP with the substrate analogue d-Arg bound shows how the enzyme binds its substrate and indicates why d-Arg is not a substrate. On the basis of this work and previous work with MppR, we propose a plausible biosynthetic scheme for l-End

    Schematic representation of RitR regulation in <i>S</i>. <i>pneumoniae</i>.

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    <p>Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is primarily produced from <i>S</i>. <i>pneumoniae</i> metabolism via pyruvate oxidase (SpxB). When present, iron must be kept out of the cell, or alternatively, stored such that it cannot react to yield Fenton chemistry, thereby causing cellular damage. RitR regulates this process by oxidation though Cys128 in high H<sub>2</sub>O<sub>2</sub> produced in aerobic environments such as the nasopharynx, which allows its open conformation and release of the DNA-binding domain (DBD) for the Regulatory Domain (RD) to interact with the Piu promoter, repressing iron uptake. Simultaneously, RitR is postulated to remediate iron toxicity through activation of DNA repair and iron sequestration [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref025" target="_blank">25</a>]. Conversely, when H<sub>2</sub>O<sub>2</sub> concentrations are low, RitR stays in its inactive form, where the interaction of the RD with the DBD prevents its binding to the Piu regulatory region, ultimately allowing for more iron to enter the cell. The potential oxidation states of RitR (SO<sup>-</sup>, SO<sup>2-</sup>, SO<sup>3-</sup>) and their regulatory consequences remain enigmatic.</p

    Analysis of RitR mutations.

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    <p>(<b>a</b>) Cartoon representation of the RitR<sub>ALR</sub> atomic structure depicting important Gate residues (shown in green), the conserved Tyr100 and Asp81 Y/T-coupling residues (shown in orange) and acidic triad residues (shown in cyan). (<b>b</b>) SEC of RitR<sub>FL</sub> variants (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004795#ppat.1004795.s006" target="_blank">S6 Fig</a> for protein purity). mAU, milli Absorbance Units; WT, Wild-type RitR<sub>FL</sub> protein; D, Dimeric form of RitR<sub>FL</sub>; M, Monomeric form of RitR<sub>FL</sub>. (<b>c</b>) EMSA shifts of RitR<sub>FL</sub> variants in the presence of HEX-labeled BS2 33-mer double-stranded DNA oligomer at 0, 0.22, 0.66, 2.2 and 6.6 μM concentrations (left to right). P, Hex-BS2 DNA Probe; C, RitR-(HEX-BS2 DNA) shifted Complex. (<b>d</b>) EMSA quantification of RitR<sub>FL</sub> variants (2.2 μM) binding to HEX-BS2 DNA. Values represent mean +/- SEM, n = 3. (<b>e</b>) Effect of RitR mutations on Piu promoter activity as measured by β-galactosidase levels (in Miller units). One asterisk indicates a P value of <0.05, and two asterisks a P value of <0.01 as determined by Student’s t-test. Error bars represent mean +/- SEM. t-test comparisons were made using the Y100A mutant as a reference.</p

    Cys-ALRs.

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    <p>(<b>a</b>) Alignment of the 26 extracted ALR domains that contain a cysteine residue in place of the typical phosphorylated Asp seen in canonical REC domain sequences (colored yellow). For comparison, Asp-containing REC domains VanR, OmpR, PhoB, CiaR and CovR were included and their conserved phosphorylatable Asp residue (colored blue). The ALR sequences were imported in FASTA format into Clustal X 2.1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004795#ppat.1004795.ref082" target="_blank">82</a>]. The alignment was then uploaded into MacBoxShade 2.15 (Institute of Animal Health, Pirbright, UK) for visual representation. (<b>b</b>) Phylogenetic tree of Cys-ALRs shown in (a). Related clades are grouped by color and a schematic representation of their domain architecture is shown on the right. Posterior probabilities are shown at the branch points. The circle with a “C” or “D” indicates a Cys or Asp amino acid, respectively, located at the phospho-Asp position. Domain architecture abbreviations are as follows: REC, receiver; HK, histidine kinase; HTH, helix-turn-helix; wHTH, winged helix-turn-helix; DsbA, bacterial disulfide oxidoreductase; GGDEF, cyclic-di-GMP; EAL, diguanylate phosphodiesterase; HDOD and HD5, phosphohydrolase; HTH-luxR, luxR family of bacterial transcription factors; MYSc, myocin domain; DUF, domain of unknown function. The alignment was generated using Clustal X 2.1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004795#ppat.1004795.ref082" target="_blank">82</a>] and uploaded for phylogenetic display into Archaeopteryx [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004795#ppat.1004795.ref083" target="_blank">83</a>].</p

    Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

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    The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function

    <i>Streptomyces wadayamensis</i> MppP is a PLP-Dependent Oxidase, Not an Oxygenase

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    The PLP-dependent l-arginine hydroxylase/deaminase MppP from <i>Streptomyces wadayamensis</i> (SwMppP) is involved in the biosynthesis of l-enduracididine, a nonproteinogenic amino acid found in several nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of l-arginine to form a mixture of 2-oxo-4­(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady-state kinetics analysis in the presence and absence of catalase shows that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo-4­(S)-hydroxy-5-guanidinovaleric acid produced, two molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either <sup>[18]</sup>O<sub>2</sub> or H<sub>2</sub><sup>[18]</sup>O and analyzing the products by ESI-MS. Incorporation of <sup>[18]</sup>O was only observed when the reaction was performed in H<sub>2</sub><sup>[18]</sup>O. Crystal structures of SwMppP with l-arginine, 2-oxo-4­(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at resolutions of 2.2, 1.9. and 1.8 Å, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of N-terminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of l-arginine

    Structure of the ‘reduced’ RitR C128S.

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    <p>(<b>A</b>) RitR Clustal Omega annotated alignment of RitR and two other full-length response regulators with available structures (MtrA from <i>Mycobacterium tuberculosis</i>, PDB ID 2GWR, and Rra from <i>Deinococcus radiodurans</i>, PDB ID 3Q9S). Identity is denoted by an asterisk and similarity by dots/colons. Secondary structure depicted above the sequences is color coordinated with the 3D models presented in <i>B-D</i>. The reactive Cys128 position is shaded in yellow, and the position which normally contains the phosphorylated Asp residue shaded in blue (note in RitR it is an Asn instead). (<b>B</b>) Ribbon diagram of the full-length, monomeric ('reduced' / inactive) RitR<sub>C128S</sub>. Ser128 (Cys128 coordinate) is labeled, colored yellow and appears in ball-and-stick format. The α4-β5-α5 face of the REC domain used by canonical response regulators for dimerization is shown in green. The remainder of the REC domain is blue. The DNA-binding domain is gold, save for the recognition helix (red) and the trans-activation loop (magenta) that interacts with RNA polymerase to direct transcription [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref048" target="_blank">48</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref049" target="_blank">49</a>]. ALR, Aspartate-less receiver domain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref030" target="_blank">30</a>]; DBD, DNA-binding domain. (<b>C</b>) Close-up of the RitR DBD-REC interface shown with the same coloring. The residues comprising the interface are shown as ball-and-stick. (<b>D</b>) Close-up of the Ser128 (Cys128 coordinate) interactions with neighboring residues and water molecules. Dotted lines denote predicted electrostatic interactions. Oxygen atoms are shown in red, nitrogens in blue and water molecules as light blue circles. Images were created using MOLSCRIPT and POVRay [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007052#ppat.1007052.ref095" target="_blank">95</a>].</p
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