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

ABSTRACT: The Cori ester ?-d-glucose 1-phosphate (?Glc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes ?Glc 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 ?Glc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic ?Glc 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 ?Glc 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 ?Glc 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 ?Glc 1-P. By examining phosphoryl transfer from ?Glc 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 ?Glc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over ?Glc 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 ?Glc 1-P.

SUBMITTER: Wildberger P 

PROVIDER: S-EPMC4325146 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature