title: Phosphoryl transfer from α-d-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types.
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.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Graz, Austria.
authors.forename: Patricia
authors.initials: P
authors.lastname: Wildberger
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Graz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pfeiffer
authors.affiliation: Institute of Organic Chemistry, University of Vienna, Vienna, Austria.
authors.forename: Lothar
authors.initials: L
authors.lastname: Brecker
authors.affiliation: Institute of Molecular Biosciences, University of Graz, NAWI Graz, Graz, Austria Omics Center Graz, BioTechMed Graz, Graz, Austria.
authors.forename: Gerald N
authors.initials: GN
authors.lastname: Rechberger
authors.affiliation: Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz, Graz, Austria Omics Center Graz, BioTechMed Graz, Graz, Austria.
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
authors.affiliation: 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.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005958:Glucosephosphates / Q000378:metabolism*; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010758:Phosphorus / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D005958:Glucosephosphates; D010758:Phosphorus; C031590:glucose-1-phosphate; C000602300:Had13 protein, E coli; C062887:agp protein, E coli; D010744:Phosphoric Monoester Hydrolases; C022700:sugar-phosphatase