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Ribose utilization by the human commensal Bifidobacterium breve UCC2003.

Pokusaeva K, Neves AR, Zomer A, O'Connell-Motherway M, MacSharry J, Curley P, Fitzgerald GF, van Sinderen D - Microb Biotechnol (2009)

Bottom Line: Growth of Bifidobacterium breve UCC2003 on ribose leads to the transcriptional induction of the rbsACBDK gene cluster.Generation and phenotypic analysis of an rbsA insertion mutant established that the rbs gene cluster is essential for ribose utilization, and that its transcription is likely regulated by a LacI-type regulator encoded by rbsR, located immediately upstream of rbsA.The rbsK gene of the rbs operon of B. breve UCC2003 was shown to specify a ribokinase (EC 2.7.1.15), which specifically directs its phosphorylating activity towards D-ribose, converting this pentose sugar to ribose-5-phosphate.

View Article: PubMed Central - PubMed

Affiliation: Alimentary Pharmabiotic Centre, Department of Microbiology, University College Cork, Western Road, Cork, Ireland.

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1H‐NMR (A) and 31P‐NMR (B) spectra of the ribokinase reaction. The sample was prepared in 2H2O as described in Experimental procedures and the spectra were acquired at 33°C. Proton and phosphorus spectra were processed with 0.1 and 1 Hz line broadenings. Rib5P, ribose‐5‐phosphate; the peaks indicated with an asterisk represent ribose. The inset (in B) shows magnification of the region between 4.49 and 4.47 ppm.
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f4: 1H‐NMR (A) and 31P‐NMR (B) spectra of the ribokinase reaction. The sample was prepared in 2H2O as described in Experimental procedures and the spectra were acquired at 33°C. Proton and phosphorus spectra were processed with 0.1 and 1 Hz line broadenings. Rib5P, ribose‐5‐phosphate; the peaks indicated with an asterisk represent ribose. The inset (in B) shows magnification of the region between 4.49 and 4.47 ppm.

Mentions: Using ribose and ATP as substrates the expected ribokinase activity of the rbsK gene product was confirmed by NMR (see Experimental procedures), which also allowed characterization of the phosphorylated product of the bifidobacterial ribokinase. The obtained 1H‐NMR spectrum of the reaction displayed two signals (5.43 and 5.27 ppm, Fig. 4A), in the region where the anomeric protons of sugars typically appear. These two resonances partially overlap those of ribose at 5.42 and 5.29 ppm corresponding to the α‐furanosic and β‐furanosic conformations, respectively (data not shown), whereas no resonances corresponding to the pyranosic forms were detected (β‐pyranoribose, 4.88 ppm; α‐pyranoribose, 4.82 ppm). The proton‐decoupled 31P‐NMR spectrum showed two partially overlapping resonances (4.49 and 4.47 ppm, Fig. 4B, magnification) in the phosphomonoester region. These resonances appeared as triplets in a proton‐coupled 31P‐NMR spectrum, indicating that the phosphate group is in the vicinity of two protons as in carbon 5 (CH2). Altogether, these data led us conclude that the product of the ribokinase reaction was ribose‐5‐phosphate, which was confirmed by comparing 1H‐ and 31P‐spectra with those derived from commercially obtained ribose‐5‐phosphate.


Ribose utilization by the human commensal Bifidobacterium breve UCC2003.

Pokusaeva K, Neves AR, Zomer A, O'Connell-Motherway M, MacSharry J, Curley P, Fitzgerald GF, van Sinderen D - Microb Biotechnol (2009)

1H‐NMR (A) and 31P‐NMR (B) spectra of the ribokinase reaction. The sample was prepared in 2H2O as described in Experimental procedures and the spectra were acquired at 33°C. Proton and phosphorus spectra were processed with 0.1 and 1 Hz line broadenings. Rib5P, ribose‐5‐phosphate; the peaks indicated with an asterisk represent ribose. The inset (in B) shows magnification of the region between 4.49 and 4.47 ppm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815373&req=5

f4: 1H‐NMR (A) and 31P‐NMR (B) spectra of the ribokinase reaction. The sample was prepared in 2H2O as described in Experimental procedures and the spectra were acquired at 33°C. Proton and phosphorus spectra were processed with 0.1 and 1 Hz line broadenings. Rib5P, ribose‐5‐phosphate; the peaks indicated with an asterisk represent ribose. The inset (in B) shows magnification of the region between 4.49 and 4.47 ppm.
Mentions: Using ribose and ATP as substrates the expected ribokinase activity of the rbsK gene product was confirmed by NMR (see Experimental procedures), which also allowed characterization of the phosphorylated product of the bifidobacterial ribokinase. The obtained 1H‐NMR spectrum of the reaction displayed two signals (5.43 and 5.27 ppm, Fig. 4A), in the region where the anomeric protons of sugars typically appear. These two resonances partially overlap those of ribose at 5.42 and 5.29 ppm corresponding to the α‐furanosic and β‐furanosic conformations, respectively (data not shown), whereas no resonances corresponding to the pyranosic forms were detected (β‐pyranoribose, 4.88 ppm; α‐pyranoribose, 4.82 ppm). The proton‐decoupled 31P‐NMR spectrum showed two partially overlapping resonances (4.49 and 4.47 ppm, Fig. 4B, magnification) in the phosphomonoester region. These resonances appeared as triplets in a proton‐coupled 31P‐NMR spectrum, indicating that the phosphate group is in the vicinity of two protons as in carbon 5 (CH2). Altogether, these data led us conclude that the product of the ribokinase reaction was ribose‐5‐phosphate, which was confirmed by comparing 1H‐ and 31P‐spectra with those derived from commercially obtained ribose‐5‐phosphate.

Bottom Line: Growth of Bifidobacterium breve UCC2003 on ribose leads to the transcriptional induction of the rbsACBDK gene cluster.Generation and phenotypic analysis of an rbsA insertion mutant established that the rbs gene cluster is essential for ribose utilization, and that its transcription is likely regulated by a LacI-type regulator encoded by rbsR, located immediately upstream of rbsA.The rbsK gene of the rbs operon of B. breve UCC2003 was shown to specify a ribokinase (EC 2.7.1.15), which specifically directs its phosphorylating activity towards D-ribose, converting this pentose sugar to ribose-5-phosphate.

View Article: PubMed Central - PubMed

Affiliation: Alimentary Pharmabiotic Centre, Department of Microbiology, University College Cork, Western Road, Cork, Ireland.

Show MeSH