Ligand binding specificity of RutR, a member of the TetR family of transcription regulators in<i>Escherichia coli</i>

Minh, Phu Nguyen Le; Cima, Sergio de; Bervoets, Indra; Maes, Dominique; Rubio, Vicente; Charlier, Daniël

doi:10.1016/j.fob.2015.01.002

Cited by 17 publications

(25 citation statements)

References 39 publications

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“…2) that bears a RUT box (-177 to -192) (Shimada et al 2007; Nguyen Ple et al 2010). In contrast to previous work (Shimada et al 2007), structural work, modeling and mutant studies established that uracil but not thymine is the physiologically relevant ligand that abolishes DNA binding (Nguyen Le Minh et al 2015). As the PepA and RutR binding sites overlap, competition in the binding of PepA and RutR has been proposed but not proven yet (Shimada et al 2007).…”

Section: Regulation Of Transcription Initiation At P1contrasting

confidence: 66%

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

Charlier

Minh

Roovers³

2018

Amino Acids

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In all organisms, carbamoylphosphate (CP) is a precursor common to the synthesis of arginine and pyrimidines. In Escherichia coli and most other Gram-negative bacteria, CP is produced by a single enzyme, carbamoylphosphate synthase (CPSase), encoded by the carAB operon. This particular situation poses a question of basic physiological interest: what are the metabolic controls coordinating the synthesis and distribution of this high-energy substance in view of the needs of both pathways? The study of the mechanisms has revealed unexpected moonlighting gene regulatory activities of enzymes and functional links between mechanisms as diverse as gene regulation and site-specific DNA recombination. At the level of enzyme production, various regulatory mechanisms were found to cooperate in a particularly intricate transcriptional control of a pair of tandem promoters. Transcription initiation is modulated by an interplay of several allosteric DNA-binding transcription factors using effector molecules from three different pathways (arginine, pyrimidines, purines), nucleoid-associated factors (NAPs), trigger enzymes (enzymes with a second unlinked gene regulatory function), DNA remodeling (bending and wrapping), UTP-dependent reiterative transcription initiation, and stringent control by the alarmone ppGpp. At the enzyme level, CPSase activity is tightly controlled by allosteric effectors originating from different pathways: an inhibitor (UMP) and two activators (ornithine and IMP) that antagonize the inhibitory effect of UMP. Furthermore, it is worth noticing that all reaction intermediates in the production of CP are extremely reactive and unstable, and protected by tunneling through a 96 Å long internal channel.

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Section: Regulation Of Transcription Initiation At P1contrasting

confidence: 66%

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

Charlier

Minh

Roovers³

2018

Amino Acids

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“…For further details, see . (C) Superposition (cartoon representation) of the LBD domain of apo‐native AmtR (in red) on the dimer of the same domain of RutR (; PDB file 4X1E), with one subunit in blue and the other in grey, showing how helix 8* of RutR (in lighter blue) is projected on the other subunit, whereas the corresponding α8–α9 loop of AmtR (in pink) recedes over its own subunit. (D) Detail of the superposition in (C) showing the subunit of RutR (coloured grey) in surface representation, to illustrate the cavity obliterated in RutR by helix 8* from the adjacent subunit, but which is fully exposed in AmtR.…”

Section: Resultsmentioning

confidence: 99%

Structure of AmtR, the global nitrogen regulator of Corynebacterium glutamicum, in free and DNA‐bound forms

Palanca

Rubio

2016

The FEBS Journal

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Corynebacterium glutamicum is a bacterium used for industrial amino acid production, and understanding its metabolic pathway regulation is of high biotechnological interest. Here, we report crystal structures of AmtR, the global nitrogen regulator of C. glutamicum, in apo (2.25‐Å and 2.65‐Å resolution) and DNA‐bound (3‐Å resolution) forms. These structures reveal an all‐α homodimeric TetR family regulator composed of a helix–turn–helix‐hosting N‐terminal DNA‐binding domain and a C‐terminal dimerization domain. AmtR has several unique structural features that appear to be invariant among AmtR proteins, which may be related to its regulation by the nitrogen‐sensing trimeric protein GlnK rather than by small‐molecule effectors. As compared with other TetR family members, AmtR has an extra C‐terminal helix, a large extended external loop that resembles the flexible tranducer T‐loop of GlnK in sequence, and a large open cavity towards the intersubunit region that changes shape upon DNA binding. The marked kinking of helix 4 decreases in the DNA‐bound form. The binding of one AmtR dimer to its DNA operator involves not only the insertion of helices 3 and 3′ in adjacent turns of the double‐helix major groove, but also the anchoring of 19‐residue, arginine‐rich and proline‐rich N‐terminal extensions to two external minor grooves. Electrophoretic mobility shift assays with a deletion mutant reveal that the 19‐residue extension is crucial for AmtR binding to DNA. N‐extension anchoring explains the flanking by AT sequences of the recognized target DNA sequence core. The significance of these findings for the entire TetR family of regulators and for GlnK regulation of AmtR is discussed. Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank, http://www.pdb.org [PDB ID codes http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5DXZ (native AmtR), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5DY1 (SeMet–AmtR), and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5DY0 (AmtR·DNA)].

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“…The rut operon is activated at nitrogen-limiting conditions by NtrC, which also activates transcription of a multitude of transporters for various nitrogencontaining compounds (Zimmer et al, 2000). In addition, the rut operon is subject to transcriptional repression by RutR which is relieved by uracil (Nguyen Le Minh et al, 2015). RutR is a global regulator which represses the degradation of pyrimidines (rut operon) and purines (gcl operon) and enhances the de novo synthesis of pyrimidines thus maintaining metabolic balance between pyrimidines and purines (Shimada et al, 2007(Shimada et al, , 2008Nguyen Le Minh et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the rut operon is subject to transcriptional repression by RutR which is relieved by uracil (Nguyen Le Minh et al, 2015). RutR is a global regulator which represses the degradation of pyrimidines (rut operon) and purines (gcl operon) and enhances the de novo synthesis of pyrimidines thus maintaining metabolic balance between pyrimidines and purines (Shimada et al, 2007(Shimada et al, , 2008Nguyen Le Minh et al, 2015). The transporters encoded in the catabolic rut and gcl operons (RutG and YbbW respectively) may also confer to this metabolic balance.…”

Section: Discussionmentioning

confidence: 99%

“…Escherichia coli and other proteobacteria use the Rut (pyrimidine utilization) pathway, encoded in the rut operon (Loh et al, 2006;Osterman, 2006;Kim et al, 2010;Parales and Ingraham, 2010). Transcription of this operon is controlled by the RutR regulator (Shimada et al, 2007(Shimada et al, , 2008Nguyen Le Minh et al, 2015) and, as a superimposed control, by the nitrogen regulatory protein C (NtrC) (Zimmer et al, 2000). The Rut pathway starts with oxidative cleavage of the uracil ring by a pyrimidine oxygenase, leads to release of the two pyrimidine nitrogens as ammonium and produces a number of toxic intermediates (peracids, ureidoacrylate, aminoacrylate, malonic semialdehyde) which are converted to 3-hydroxypropionic acid that is excreted.…”

Section: Introductionmentioning

confidence: 99%

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Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli

Botou

Lazou

Παπακώστας

et al. 2018

Molecular Microbiology

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The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase-Ascorbate Transporter (NAT) or Nucleobase-Cation Symporter-2 (NCS2) family and represents a well-defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyrimidine utilization) operon. Using amplified expression in E. coli K-12, we show that RutG is a high-affinity permease for uracil, thymine and, at low efficiency, xanthine and recognizes also 5-fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5-fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site-directed replacement of this Phe with Ala in the three uracil-specific homologs allows high-affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates and provide the bacterial cell with a range of catabolizable nucleobases.

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Ligand binding specificity of RutR, a member of the TetR family of transcription regulators inEscherichia coli

Abstract: Graphical abstract

Cited by 17 publications

References 39 publications

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

Structure of AmtR, the global nitrogen regulator of Corynebacterium glutamicum, in free and DNA‐bound forms

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli

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