Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types
ABSTRACTThe 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-Pfor flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases fromEscherichia coliwere characterized in this study for utilization of αGlc 1-Pas a phosphoryl donor substrate. Theagpgene encodes a periplasmic αGlc 1-Pphosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (inE. coliOrigami B) gave a functional enzyme preparation (kcatfor phosphoryl transfer from αGlc 1-Pto water, 40 s−1) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys189and Cys195. Enzymatic phosphoryl transfer from αGlc 1-Pto water in H218O solvent proceeded with complete18O 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, andd-glucose 6-phosphate was converted with akcatsimilar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-Pto an acceptor substrate other than water (d-fructose ord-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-Pmainly for phosphoryl transfer to water. By applyingd-fructose in 10-fold molar excess over αGlc 1-P(20 mM), enzymatic conversion furnishedd-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.