Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum

Liu, Yi; Li, Wei; Wei, Yaozhu; Jiang, Yindi; Tan, Xiangshi

doi:10.1039/c3mt00163f

Cited by 7 publications

(5 citation statements)

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“…For example, the binding of Mn 2+ to the TroR enzyme was also reported as an entropy-driven process. 25 The fitting of the experimental data revealed the binding stoichiometry of Mn 2+ /enzyme (wt or C139S) in the range of 5-6, which is in agreement with the HR-ICP-MS results. The enzymes studied here had a His-tag (hexa histidine-tag) for purification reasons, and the His-tag is well known to bind not only Ni 2+ cations, but other cations as well.…”

Section: Importance Of the Conserved Cys Residues For Enzyme Activitysupporting

confidence: 85%

The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability

Smolko

Šupljika

Martinčić

et al. 2016

Phys. Chem. Chem. Phys.

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Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate.

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Section: Importance Of the Conserved Cys Residues For Enzyme Activitysupporting

confidence: 85%

The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability

Smolko

Šupljika

Martinčić

et al. 2016

Phys. Chem. Chem. Phys.

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“…T. pallidum troABCD was shown to be negatively regulated by a zincresponsive transcriptional regulator, TroR, a DtxR-like repressor (29). However, a later study showed that T. pallidum TroR is a manganese-dependent rather than a zinc-dependent regulator (31). Unlike many other bacteria, T. pallidum does not contain Zur, and the regulator of T. pallidum znuABC has not been reported.…”

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confidence: 96%

Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions

Chaoprasid

Dokpikul

Johnrod

et al. 2016

Appl Environ Microbiol

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Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens. However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions. IMPORTANCEBoth host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of hostpathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which ZinT functions in concert with the high-affinity zinc importer TroCBA. Zinc is an essential metal for bacteria because it is required for the functions of many enzymes and proteins (1, 2). However, zinc overload is toxic to cells (3-7). Bacteria have mechanisms to maintain zinc homeostasis via the coordinated response of genes involved in zinc uptake, efflux, and storage (8-13). The zinc uptake regulator Zur is a transcriptional regulator belonging to the Fur family and functions as a repressor of zinc uptake genes, including znuABC and zinT (10). To prevent excessive amounts of zinc in cells under high-zinc conditions, Zur uses Zn 2ϩ as its cofactor to bind to a conserved AT-rich sequence, called the Zur box (14), found in the promoter region of the zinc uptake genes, leading to inhibition of gene expression (15)(16)(17). ZnuA is a periplasmic protein that binds zinc and transfers it to the membrane permease ZnuB and the ATPase ZnuC (15). The ZinT protein is a periplasmic zinc-binding protein (18-21) that has been shown to directly interact with and assist ZnuABC i...

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“…The ferric uptake regulator Fur and zinc uptake regulator Zur constitute the majority of the Fur family metalloregulators, being widely distributed in diverse lineages of Bacteria but not Archaea, whereas the manganese-and nickel-specific regulators Mur and Nur were found in only some bacterial lineages (9,10). Manganese-responsive DtxR family regulators were studied in many bacteria, including MntR in Escherichia coli (11), Bacillus subtilis (12), Staphylococcus aureus (13), Corynebacterium diphtheria (14), and C. glutamicum (15,16), ScaR in Streptococcus gordonii (17), and TroR in Treponema pallidum (18), where they mostly control genes for manganese uptake transporters. In contrast, iron-responsive TFs from the DtxR family were found only in Actinobacteria and include two experimentally studied TFs, the diphtheria toxin repressor DtxR in Corynebacterium diphtheriae (19) and the iron-dependent regulator IdeR in Mycobacterium tuberculosis (20,21), which control large networks of genes involved in iron homeostasis and other cellular functions, such as the toxin gene in C. diphtheriae.…”

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confidence: 99%

Comparative Genomics of DtxR Family Regulons for Metal Homeostasis in Archaea

Leyn

Rodionov

2014

J. Bacteriol.

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bThe DtxR family consists of metal-dependent transcription factors (DtxR-TFs) that regulate the expression of genes involved in metal homeostasis in the cell. The majority of characterized DtxR-TFs belong to Bacteria. In the current work, we applied a comparative genomics approach to predict DNA-binding sites and reconstruct regulons for DtxR-TFs in Archaea. As a result, we inferred 575 candidate binding sites for 139 DtxR-TFs in 77 genomes from 15 taxonomic orders. Novel DNA motifs of archaeal DtxR-TFs that have a common palindromic structure were classified into 10 distinct groups. By combining functional regulon reconstructions with phylogenetic analysis, we selected 28 DtxR-TF clades and assigned them metal specificities and regulator names. The reconstructed FetR (ferrous iron), MntR (manganese), and ZntR (zinc) regulons largely contain known or putative metal uptake transporters from the FeoAB, NRAMP, ZIP, and TroA families. A novel family of putative iron transporters (named Irt), including multiple FetR-regulated paralogs, was identified in iron-oxidizing Archaea from the Sulfolobales order. The reconstructed DtxR-TF regulons were reconciled with available transcriptomics data in Archaeoglobus, Halobacterium, and Thermococcus spp.T ransition metals, including iron, manganese, and zinc, are responsible for a diverse array of biochemical reactions and other biological functions in prokaryotes. The particular properties of ferrous iron (Fe 2ϩ ) and ferric iron (Fe 3ϩ ) ions make them widely used redox-sensing elements in many metalloenzymes, from heme-containing cytochromes to Fe-S proteins. Manganese is used in free radical-detoxifying enzymes, including superoxide dismutase and catalase and some other enzymes. Zinc was found in many proteins, including enzymes of nucleic acid metabolism and ribosomal proteins, where it plays a role in both catalysis and protein structure. Many transition metals are taken up by specific transport systems, including FeoAB family transporters for ferrous iron (1, 2), Fbp-type ATP-binding cassette (ABC) transporters for ferric iron (3), Znu family ABC transporters for zinc (4), and NRAMP family MntR transporters for manganese (5). The precise maintenance of metal ion homeostasis, including metal uptake transporters, is vital for cells that have to balance between supplying enzymes with metal ion cofactors and decreasing the harmful effects of heavy metals (6, 7).The regulation of gene expression by specific binding of transcription factors (TFs) to their DNA sites in response to a cellular signal (e.g., specific metal ions) is a common regulatory mechanism in microorganisms. Iron, manganese, and zinc homeostasis genes in prokaryotes are controlled by TFs from two major families of metalloregulators, Fur and DtxR (8). The ferric uptake regulator Fur and zinc uptake regulator Zur constitute the majority of the Fur family metalloregulators, being widely distributed in diverse lineages of Bacteria but not Archaea, whereas the manganese-and nickel-specific regulators Mur and Nur...

show abstract

Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum

Cited by 7 publications

References 50 publications

The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability

The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability

Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions

Comparative Genomics of DtxR Family Regulons for Metal Homeostasis in Archaea

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