Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

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 ( k cat 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 H 2 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 k cat 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 .