Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL

Johanne M Jensen; Heidi A Ernst; Xiaole Wang; Helle Hald; Amarah C Ditta; Fouzia Ismat; Moazur Rahman; Osman Mirza

doi:10.2174/092986612799363109

Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL

Johanne M Jensen, Heidi A Ernst, Xiaole Wang, Helle Hald, Amarah C Ditta, Fouzia Ismat, Moazur Rahman, Osman Mirza

15 Citations (Scopus)

Abstract

Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC ₅₀ values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ∼0.1 to ∼1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC ₅₀ increased to ∼2.0 and >10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.

Original language	English
Journal	Protein and Peptide Letters
Volume	19
Issue number	3
Pages (from-to)	282-287
ISSN	0929-8665
DOIs	https://doi.org/10.2174/092986612799363109
Publication status	Published - Mar 2012

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@article{5182c0b9a72e48ebbc7b20af496a034c,

title = "Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL",

abstract = "Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC 50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ∼0.1 to ∼1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC 50 increased to ∼2.0 and >10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.",

author = "Jensen, {Johanne M} and Ernst, {Heidi A} and Xiaole Wang and Helle Hald and Ditta, {Amarah C} and Fouzia Ismat and Moazur Rahman and Osman Mirza",

year = "2012",

month = mar,

doi = "10.2174/092986612799363109",

language = "English",

volume = "19",

pages = "282--287",

journal = "Protein and Peptide Letters",

issn = "0929-8665",

publisher = "Bentham Science Publishers",

number = "3",

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TY - JOUR

T1 - Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL

AU - Jensen, Johanne M

AU - Ernst, Heidi A

AU - Wang, Xiaole

AU - Hald, Helle

AU - Ditta, Amarah C

AU - Ismat, Fouzia

AU - Rahman, Moazur

AU - Mirza, Osman

PY - 2012/3

Y1 - 2012/3

N2 - Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC 50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ∼0.1 to ∼1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC 50 increased to ∼2.0 and >10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.

AB - Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC 50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ∼0.1 to ∼1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC 50 increased to ∼2.0 and >10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.

U2 - 10.2174/092986612799363109

DO - 10.2174/092986612799363109

M3 - Journal article

C2 - 21933132

SN - 0929-8665

VL - 19

SP - 282

EP - 287

JO - Protein and Peptide Letters

JF - Protein and Peptide Letters

IS - 3

ER -

Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL

Abstract

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