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Enzymatic synthesis of nucleobase-modified UDP-sugars: scope and limitations.

Wagstaff BA, Rejzek M, Pesnot T, Tedaldi LM, Caputi L, O'Neill EC, Benini S, Wagner GK, Field RA - Carbohydr. Res. (2014)

Bottom Line: Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield.In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields.Attempts to effect 4"-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4"-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

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Reverse action of GalU in the presence of excess 10 mM PPi. (A) Conversion of 5-iodo-UDP-Glc (2b) into Glc-1-P. (B) The same as A, but in the presence of excess (5 equiv) 5-iodo-UDP-Gal (1b).
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f0030: Reverse action of GalU in the presence of excess 10 mM PPi. (A) Conversion of 5-iodo-UDP-Glc (2b) into Glc-1-P. (B) The same as A, but in the presence of excess (5 equiv) 5-iodo-UDP-Gal (1b).

Mentions: The lack of GalU-mediated conversion of Gal-1-P with 5-iodo-UTP was somewhat unexpected and warranted further analysis. First, it was shown that in the presence of a high concentration of inorganic pyrophosphate (PPi), GalU can perform the reverse conversion from 5-iodo-UDP-Glc (2b) to Glc-1-P and 5-iodo-UTP (5b). The conversion was complete, as judged by 1H NMR [anomeric proton resonances (dd) were used as diagnostic peaks], within 10 min with 10 mM PPi (Fig. 6). When 5-iodo-UDP-Gal (1b) was subjected to analogous conditions no conversion was observed even after incubation for 60 min (data not shown). To see whether the lack of conversion of the galacto-configured substrates was down to lack of binding or to non-productive binding of the substrates, inhibition experiments employing 5 equiv of 5-iodo-UDP-Gal (1b), 1 equiv of 5-iodo-UDP-Glc (2b) and excess PPi were conducted. By 1H NMR, 5-iodo-UDP-Glc (2b) was fully converted into Glc-1-P and 5-iodo-UTP (5b) within 10 min as in the no inhibitor control reaction (Fig. 6). No conversion 5-iodo-UDP-Gal (1b) was detected, which suggests that 5-iodo-UDP-Gal (1b) does not bind to the active site of GalU. A competition experiment was designed to show whether a large excess of Gal-1-P (5 equiv) can outcompete the natural acceptor Glc-1-P (1 equiv) in a GalU mediated conversion of 5-iodo-UTP (5b) (1 equiv) into 5-iodo-UDP-sugar. 1H NMR spectra showed that only 5-iodo-UDP-Glc (2b) was formed and no trace of 5-iodo-UDP-Gal (1b) was detected, even after 120 min (not shown).


Enzymatic synthesis of nucleobase-modified UDP-sugars: scope and limitations.

Wagstaff BA, Rejzek M, Pesnot T, Tedaldi LM, Caputi L, O'Neill EC, Benini S, Wagner GK, Field RA - Carbohydr. Res. (2014)

Reverse action of GalU in the presence of excess 10 mM PPi. (A) Conversion of 5-iodo-UDP-Glc (2b) into Glc-1-P. (B) The same as A, but in the presence of excess (5 equiv) 5-iodo-UDP-Gal (1b).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340641&req=5

f0030: Reverse action of GalU in the presence of excess 10 mM PPi. (A) Conversion of 5-iodo-UDP-Glc (2b) into Glc-1-P. (B) The same as A, but in the presence of excess (5 equiv) 5-iodo-UDP-Gal (1b).
Mentions: The lack of GalU-mediated conversion of Gal-1-P with 5-iodo-UTP was somewhat unexpected and warranted further analysis. First, it was shown that in the presence of a high concentration of inorganic pyrophosphate (PPi), GalU can perform the reverse conversion from 5-iodo-UDP-Glc (2b) to Glc-1-P and 5-iodo-UTP (5b). The conversion was complete, as judged by 1H NMR [anomeric proton resonances (dd) were used as diagnostic peaks], within 10 min with 10 mM PPi (Fig. 6). When 5-iodo-UDP-Gal (1b) was subjected to analogous conditions no conversion was observed even after incubation for 60 min (data not shown). To see whether the lack of conversion of the galacto-configured substrates was down to lack of binding or to non-productive binding of the substrates, inhibition experiments employing 5 equiv of 5-iodo-UDP-Gal (1b), 1 equiv of 5-iodo-UDP-Glc (2b) and excess PPi were conducted. By 1H NMR, 5-iodo-UDP-Glc (2b) was fully converted into Glc-1-P and 5-iodo-UTP (5b) within 10 min as in the no inhibitor control reaction (Fig. 6). No conversion 5-iodo-UDP-Gal (1b) was detected, which suggests that 5-iodo-UDP-Gal (1b) does not bind to the active site of GalU. A competition experiment was designed to show whether a large excess of Gal-1-P (5 equiv) can outcompete the natural acceptor Glc-1-P (1 equiv) in a GalU mediated conversion of 5-iodo-UTP (5b) (1 equiv) into 5-iodo-UDP-sugar. 1H NMR spectra showed that only 5-iodo-UDP-Glc (2b) was formed and no trace of 5-iodo-UDP-Gal (1b) was detected, even after 120 min (not shown).

Bottom Line: Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield.In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields.Attempts to effect 4"-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4"-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Show MeSH