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 Cys189 and Cys195. Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 18O solvent proceeded with complete 18O 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.
Originalsprache | englisch |
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Seiten (von - bis) | 1559-1572 |
Seitenumfang | 14 |
Fachzeitschrift | Applied and Environmental Microbiology |
Jahrgang | 81 |
Ausgabenummer | 5 |
DOIs | |
Publikationsstatus | Veröffentlicht - 2015 |
ASJC Scopus subject areas
- Angewandte Mikrobiologie und Biotechnologie
- Lebensmittelwissenschaften
- Biotechnology
- Ökologie