A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

Kaur, Jagdeep; Olkhova, Elena; Malviya, Viveka Nand; Grell, Ernst; Michel, Hartmut

doi:10.1074/jbc.m113.510743

Cited by 13 publications

(15 citation statements)

References 42 publications

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“…With a protein molecular weight of 54 kDa, the observed elution behaviour therefore suggests that LysP exists in micellar solution as a monomer. This is consistent with a separate report concerning the oligomeric state of LysP from S. typhimurium (Kaur et al, 2014). X-ray diffraction from a LysP crystal grown in surfo at 20 C. The diffraction pattern shown was recorded on a PILATUS 6M detector with 0.978 Å wavelength X-rays at 1 oscillation and 1 s exposure, a micro-focus beam size of 10 mm and a sample-todetector distance of 450 mm.…”

Section: Expression and Purification Of Lyspsupporting

confidence: 70%

“…Isothermal titration calorimetry was used to establish that residues Lys163, Glu222 and Arg395 are important in this process. These residues are well conserved in the APC family, suggesting that LysP shares a similar transport mechanism to other APC members (Kaur et al, 2014). However, the detailed mechanism of substrate recognition, selectivity and transport have yet to be elucidated.…”

Section: Introductionmentioning

confidence: 99%

“…Sharing 70% identity with its E. coli counterpart, LysP from Pseudomonas aeruginosa has been shown to specifically transport l-lysine in liposomes using energy derived from a proton gradient (Nji et al, unpublished work). Based on homology modelling, a number of residues that impact on lysine transport by LysP from Salmonella typhimurium have been identified (Kaur et al, 2014). Isothermal titration calorimetry was used to establish that residues Lys163, Glu222 and Arg395 are important in this process.…”

Section: Introductionmentioning

confidence: 99%

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

Nji

Doyle

et al. 2014

Acta Cryst Sect F

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The prokaryotic lysine-specific permease (LysP) belongs to the amino acidpolyamine-organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of l-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal-and rod-shaped crystals were obtained in the presence of l-lysine and the l-lysine analogue l-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space group P2 1 , with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å , = 120 .

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Section: Expression and Purification Of Lyspsupporting

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

Nji

Doyle

et al. 2014

Acta Cryst Sect F

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“…For functional studies, E. coli strain KAM32 (a kind gift of Dr. Teruo Kuroda from Okayama University) was used, which lacks AcrB and YdhE transporters and thus becomes drug-hypersensitive (34). For heterologous protein production in E. coli, a modified pBAD vector (Invitrogen), pBADA2, which contains a tobacco etch virus protease cleavage site and a decahistidine tag (His tag) attached at the C terminus of the target protein, was used (35).…”

Section: Methodsmentioning

confidence: 99%

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

Nie

Grell

Malviya

et al. 2016

Journal of Biological Chemistry

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Multidrug and toxic compound extrusion (MATE) transportThe ability of all living organisms to protect themselves against toxic substances is essential for their survival. Organisms acquire this ability through evolutionary force (1). Due to the development of scientific knowledge, more and more medical drugs are being developed and applied. As a consequence, general defense mechanisms to resist antibiotics, drugs, and other toxic compounds have been evolved in bacteria (2). Being one of those defense mechanisms, the active extrusion of the toxic compounds from the cells plays an important role (3). In bacteria, the extrusion is often carried out by multidrug transporters, which are integral membrane proteins. They are classified into five distinct families: ATP-binding cassette, multidrug and toxic compound extrusion (MATE), 4 major facilitator superfamily, resistance nodulation division, and small multidrug resistance transporters (4).In 1999, the MATE family proteins were first recognized as a new group of the multidrug transporters, existing in all three domains of life (5). Most members of this family consist of a single chain of 450 -550 amino acid residues and exhibit 12 putative transmembrane helices (TMHs). Up to now, about 900 proteins have been annotated as MATE transporters on the basis of amino acid sequence similarities. Moreover, a subclassification of the MATE family has been suggested, NorM-, DinF-(DNA damage-inducible protein F), and eukaryotic subfamily (4, 6, 7). Various compounds can be recognized by NorM proteins, including dyes, fluoroquinolones, and aminoglycosides (7). As secondary active transporters, MATE proteins utilize transmembrane Na ϩ or H ϩ electrochemical gradients to extrude toxic compounds.So far atomic structures of four MATE transporters have been reported in their putative outward-facing states, in both drug-bound and/or drug-free states. NorM_VC from Vibrio cholerae and NorM_NG from Neisseria gonorrhoeae, are both Na ϩ driven, whereas PfMATE from Pyrococcus furiosus and DinF-BH from Bacillus halodurans are published to be H ϩ -dependent antiporters (8 -11). All these four proteins contain 12 TMHs with intracellular N and C termini, and possess a hydrophobic internal cavity, formed by two symmetric bundles with 6 TMHs each. The two bundles are linked by a cytoplasmic loop between the 6th and 7th TMH. All MATE proteins share about 40% sequence similarity, and they employ the same rockerswitch mechanism for substrate extrusion (12). In addition, the crystal structures have also revealed different locations of cationand substrate-binding sites, suggesting a mechanistic diversity among MATE transporters. In the structure of drug-bound NorM_NG, the binding site is located close to the membraneperiplasm interface, whereas in the structure of PfMATE, the

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“…Unlike the spectrofluorometric titration, which depends on quenching of intrinsic protein luminescence to detect binding, ITC is sensitive to all interactions of PLP with Mtm1p. In previous studies, ITC has proven useful in the investigation of substrate interactions of membrane carriers through analysis of the binding step involved in their transport processes [36]. We find that incremental addition of PLP to purified, refolded Mtm1p results in saturation behavior (Fig.…”

Section: Resultsmentioning

confidence: 63%

The Mtm1p carrier and pyridoxal 5′-phosphate cofactor trafficking in yeast mitochondria

Whittaker¹,

Penmatsa²,

Whittaker³

2015

Archives of Biochemistry and Biophysics

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Biochemical communication between the cytoplasmic and mitochondrial subsystems of the cell depends on solute carriers in the mitochondrial inner membrane that transport metabolites between the two compartments. We have expressed and purified a yeast mitochondrial carrier protein (Mtm1p, YGR257cp), originally identified as a manganese ion carrier, for biochemical characterization aimed at resolving its function. High affinity, stoichiometric pyridoxal 5′-phosphate (PLP) cofactor binding was characterized by fluorescence titration and calorimetry, and the biochemical effects of mtm1 gene deletion on yeast mitochondria were investigated. The PLP status of the mitochondrial proteome (the mitochondrial ‘PLP-ome’) was probed by immunoblot analysis of mitochondria isolated from wild type (MTM1+) and knockout (MTM1−) yeast, revealing depletion of mitochondrial PLP in the latter. A direct activity assay of the enzyme catalyzing the first committed step of heme biosynthesis, the PLP-dependent mitochondrial enzyme 5-aminolevulinate synthase, extends these results, providing a specific example of PLP cofactor limitation. Together, these experiments support a role for Mtm1p in mitochondrial PLP trafficking and highlight the link between PLP cofactor transport and iron metabolism, a remarkable illustration of metabolic integration.

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A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

Cited by 13 publications

References 42 publications

Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri

The Mtm1p carrier and pyridoxal 5′-phosphate cofactor trafficking in yeast mitochondria

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