Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intes... more Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 μm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the cultu...
The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change i... more The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrRE274Q, a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrRN316D and TyrRN316R mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrRN316D and TyrRN316R, respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrRN316D.
Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incu... more Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incubation at 4 °C, while heat-treated γ-glutamyltranspeptidase of Bacillus subtilis did not. Fluorescent spectra, CD spectra, and native polyacrylamide gel electrophoresis analysis suggested that the dimer of E. coli γ-glutamyltranspeptidase was separated into protomers by heat-treatment, but was renatured by incubation at 4 °C.
Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incu... more Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incubation at 4 °C, while heat-treated γ-glutamyltranspeptidase of Bacillus subtilis did not. Fluorescent spectra, CD spectra, and native polyacrylamide gel electrophoresis analysis suggested that the dimer of E. coli γ-glutamyltranspeptidase was separated into protomers by heat-treatment, but was renatured by incubation at 4 °C.
Escherichia coli K-12 cultured at 20 degrees C has more gamma-glutamyltranspeptidase (GGT: EC 2.3... more Escherichia coli K-12 cultured at 20 degrees C has more gamma-glutamyltranspeptidase (GGT: EC 2.3.2.2) activity than that cultured at 37 or 42 degrees C. On Western blot analysis, E. coli K-12 cells cultured at 20 degrees C produced more GGT protein than those cultured at 37 degrees C. mRNA of the GGT gene (ggt) in the cells was also measured and it was found that the level of ggt mRNA at 20 degrees C was 10-fold higher than that at 37 degrees C. When the ggt promoter was replaced by a tac promoter, GGT activity at 37 degrees C from the tac promoter was 5-fold higher than that at 37 degrees C from the ggt promoter, though there was less difference in GGT activity between both grown at 20 degrees C. The ggt mRNA at 20 degrees C was found to be more stable than that at 37 degrees C. These results suggested that the higher GGT activity in E. coli K-12 cells grown at 20 degrees C was due to a higher level of GGT protein at 20 degrees C caused by higher level of ggt mRNA at 20 degrees C because of a low-temperature dependent ggt promoter as well as the stability of ggt mRNA at 20 degrees C.
The structure of GGT [EC 2.3.2.2] from E. coli K-12 was studied at 3 A resolution by X-ray crysta... more The structure of GGT [EC 2.3.2.2] from E. coli K-12 was studied at 3 A resolution by X-ray crystallography. Initial protein phases were calculated using two kinds of Pb2+ derivatives. The phases were refined by non-crystallographic 2-fold symmetry electron density averaging combined with solvent flattening and histogram matching. The GGT molecule has overall dimensions of 60 x 50 x 40 A. There are two antiparallel beta-pleated sheets consisting of 6 and 7 beta-strands. The two beta-sheets form a wall-like structure. Twelve short alpha-helices were detected, of which the maximum length appears to be four helix turns.
Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intes... more Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 μm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the cultu...
The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change i... more The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrRE274Q, a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrRN316D and TyrRN316R mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrRN316D and TyrRN316R, respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrRN316D.
Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incu... more Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incubation at 4 °C, while heat-treated γ-glutamyltranspeptidase of Bacillus subtilis did not. Fluorescent spectra, CD spectra, and native polyacrylamide gel electrophoresis analysis suggested that the dimer of E. coli γ-glutamyltranspeptidase was separated into protomers by heat-treatment, but was renatured by incubation at 4 °C.
Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incu... more Heat-treated γ-glutamyltranspeptidase of Escherichia coli recovered enzymatic activity after incubation at 4 °C, while heat-treated γ-glutamyltranspeptidase of Bacillus subtilis did not. Fluorescent spectra, CD spectra, and native polyacrylamide gel electrophoresis analysis suggested that the dimer of E. coli γ-glutamyltranspeptidase was separated into protomers by heat-treatment, but was renatured by incubation at 4 °C.
Escherichia coli K-12 cultured at 20 degrees C has more gamma-glutamyltranspeptidase (GGT: EC 2.3... more Escherichia coli K-12 cultured at 20 degrees C has more gamma-glutamyltranspeptidase (GGT: EC 2.3.2.2) activity than that cultured at 37 or 42 degrees C. On Western blot analysis, E. coli K-12 cells cultured at 20 degrees C produced more GGT protein than those cultured at 37 degrees C. mRNA of the GGT gene (ggt) in the cells was also measured and it was found that the level of ggt mRNA at 20 degrees C was 10-fold higher than that at 37 degrees C. When the ggt promoter was replaced by a tac promoter, GGT activity at 37 degrees C from the tac promoter was 5-fold higher than that at 37 degrees C from the ggt promoter, though there was less difference in GGT activity between both grown at 20 degrees C. The ggt mRNA at 20 degrees C was found to be more stable than that at 37 degrees C. These results suggested that the higher GGT activity in E. coli K-12 cells grown at 20 degrees C was due to a higher level of GGT protein at 20 degrees C caused by higher level of ggt mRNA at 20 degrees C because of a low-temperature dependent ggt promoter as well as the stability of ggt mRNA at 20 degrees C.
The structure of GGT [EC 2.3.2.2] from E. coli K-12 was studied at 3 A resolution by X-ray crysta... more The structure of GGT [EC 2.3.2.2] from E. coli K-12 was studied at 3 A resolution by X-ray crystallography. Initial protein phases were calculated using two kinds of Pb2+ derivatives. The phases were refined by non-crystallographic 2-fold symmetry electron density averaging combined with solvent flattening and histogram matching. The GGT molecule has overall dimensions of 60 x 50 x 40 A. There are two antiparallel beta-pleated sheets consisting of 6 and 7 beta-strands. The two beta-sheets form a wall-like structure. Twelve short alpha-helices were detected, of which the maximum length appears to be four helix turns.
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Papers by Hideyuki Suzuki