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Appl Environ Microbiol. 2015 Mar;81(5):1559-72. doi: 10.1128/AEM.03314-14. Epub 2014 Dec 19.

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

Author information

1
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Graz, Austria.
2
Institute of Organic Chemistry, University of Vienna, Vienna, Austria.
3
Institute of Molecular Biosciences, University of Graz, NAWI Graz, Graz, Austria Omics Center Graz, BioTechMed Graz, Graz, Austria.
4
Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz, Graz, Austria Omics Center Graz, BioTechMed Graz, Graz, Austria.
5
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Graz, Austria Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria bernd.nidetzky@tugraz.at.

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 Cys(189) and Cys(195). Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 (18)O solvent proceeded with complete (18)O 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.

PMID:
25527541
PMCID:
PMC4325146
DOI:
10.1128/AEM.03314-14
[Indexed for MEDLINE]
Free PMC Article

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