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Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in L-lactate and sugar utilization.

Gao YG, Suzuki H, Itou H, Zhou Y, Tanaka Y, Wachi M, Watanabe N, Tanaka I, Yao M - Nucleic Acids Res. (2008)

Bottom Line: LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism.Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding.The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-A resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn(2+) binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

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Schematic diagram representing the functions of the two operons regulated by the transcriptional repressor LldR. The membrane is shown as a rectangular frame coloured grey.
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Figure 8: Schematic diagram representing the functions of the two operons regulated by the transcriptional repressor LldR. The membrane is shown as a rectangular frame coloured grey.

Mentions: The cgl2917-lldD operon induced during temperature-triggered glutamate production is essential for the utilization or re-utilization of l-lactate (10). The gene cgl2917 encodes a permease that is putatively involved in uptake of l-lactate (Figure 8), and lldD encodes LldD (10). LldD catalyses the oxidation of l-lactate to pyruvate, which can be converted into acetyl-CoA used in the tricarboxylic acid (TCA) cycle (Figure 8). In E. coli, the lld operon involved in l-lactate utilization is controlled by the two-component signal transduction system ArcB/ArcA, and its transcription is increased by l-lactate (49,50). The results of the present study indicated that the lldR gene encodes a repressor regulating the lld operon. Fructose is an important carbon source for industrial fermentation with C. glutamicum. However, regulation of fructose uptake is poorly understood. The present study revealed that the cgl1934-fruK-ptsF operon is also under the control of LldR. This operon encodes a transcriptional regulator of sugar metabolism, FruK and EIIFru (9). EIIFru is a fructose-specific permease involved in converting fructose into F-1-P across the membrane (Figure 8). Subsequently, F-1-P can be converted into F-1,6-2P to enter glycolysis, catalysed by FruK, which is indispensable for growth of C. glutamicum on fructose minimal medium (17). Due to the absence of a fructokinase gene in C. glutamicum (21), fructose from sucrose-6-P (product of membrane transport of the sucrose) can not be converted into F-6-P (Figure 8). Fructose was shown to be excreted, then re-assimilated via a fructose-specific PTS (21,51). Therefore, FruK and EIIFru may form an assisted pathway to utilize intracellular fructose. Thus, LldR plays a significant role in reutilization of both fructose and sucrose by regulating the transcription of the cgl1934-fruK-ptsF operon.Figure 8.


Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in L-lactate and sugar utilization.

Gao YG, Suzuki H, Itou H, Zhou Y, Tanaka Y, Wachi M, Watanabe N, Tanaka I, Yao M - Nucleic Acids Res. (2008)

Schematic diagram representing the functions of the two operons regulated by the transcriptional repressor LldR. The membrane is shown as a rectangular frame coloured grey.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602784&req=5

Figure 8: Schematic diagram representing the functions of the two operons regulated by the transcriptional repressor LldR. The membrane is shown as a rectangular frame coloured grey.
Mentions: The cgl2917-lldD operon induced during temperature-triggered glutamate production is essential for the utilization or re-utilization of l-lactate (10). The gene cgl2917 encodes a permease that is putatively involved in uptake of l-lactate (Figure 8), and lldD encodes LldD (10). LldD catalyses the oxidation of l-lactate to pyruvate, which can be converted into acetyl-CoA used in the tricarboxylic acid (TCA) cycle (Figure 8). In E. coli, the lld operon involved in l-lactate utilization is controlled by the two-component signal transduction system ArcB/ArcA, and its transcription is increased by l-lactate (49,50). The results of the present study indicated that the lldR gene encodes a repressor regulating the lld operon. Fructose is an important carbon source for industrial fermentation with C. glutamicum. However, regulation of fructose uptake is poorly understood. The present study revealed that the cgl1934-fruK-ptsF operon is also under the control of LldR. This operon encodes a transcriptional regulator of sugar metabolism, FruK and EIIFru (9). EIIFru is a fructose-specific permease involved in converting fructose into F-1-P across the membrane (Figure 8). Subsequently, F-1-P can be converted into F-1,6-2P to enter glycolysis, catalysed by FruK, which is indispensable for growth of C. glutamicum on fructose minimal medium (17). Due to the absence of a fructokinase gene in C. glutamicum (21), fructose from sucrose-6-P (product of membrane transport of the sucrose) can not be converted into F-6-P (Figure 8). Fructose was shown to be excreted, then re-assimilated via a fructose-specific PTS (21,51). Therefore, FruK and EIIFru may form an assisted pathway to utilize intracellular fructose. Thus, LldR plays a significant role in reutilization of both fructose and sucrose by regulating the transcription of the cgl1934-fruK-ptsF operon.Figure 8.

Bottom Line: LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism.Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding.The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.

ABSTRACT
LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-A resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn(2+) binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed.

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