Phosphoryl transfer from α-D-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types

Patricia Wildberger, Martin Pfeiffer, Lothar Brecker, Gerald N. Rechberger, Ruth Birner-Gruenberger, Bernd Nidetzky

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The Cori ester α-D-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s-1) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys189 and Cys195. Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 18O solvent proceeded with complete 18O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and D-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (D-fructose or D-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying D-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished D-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P.

LanguageEnglish
Pages1559-1572
Number of pages14
JournalApplied and environmental microbiology
Volume81
Issue number5
DOIs
StatusPublished - 2015

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glucose 1-phosphate
Sugar Phosphates
sugar phosphates
phosphatase
sugar
glucose
phosphate
Escherichia coli
Glucose
protein
Proteins
proteins
fructose
Water
Fructose
Phosphoric Monoester Hydrolases
substrate
Cats
sugar-phosphatase
glucose-1-phosphate

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

Phosphoryl transfer from α-D-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types. / Wildberger, Patricia; Pfeiffer, Martin; Brecker, Lothar; Rechberger, Gerald N.; Birner-Gruenberger, Ruth; Nidetzky, Bernd.

In: Applied and environmental microbiology, Vol. 81, No. 5, 2015, p. 1559-1572.

Research output: Contribution to journalArticleResearchpeer-review

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abstract = "The Cori ester α-D-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s-1) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys189 and Cys195. Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 18O solvent proceeded with complete 18O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and D-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (D-fructose or D-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying D-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished D-fructose 1-phosphate as the main product in a 55{\%} overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P.",
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