Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 81, Issue 5, Pages 1559-1572Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.03314-14
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Funding
- Austrian Science Funds FWF [L586-B03]
- Austrian Science Fund (FWF) [L586] Funding Source: Austrian Science Fund (FWF)
- Austrian Science Fund (FWF) [L 586, W 901] Funding Source: researchfish
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The Cori ester alpha-D-glucose 1-phosphate (alpha Glc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes alpha 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 alpha Glc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic alpha 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 (k(cat) for phosphoryl transfer from alpha 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 alpha Glc 1-P to water in (H2O)-O-18 solvent proceeded with complete O-18 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 alpha Glc 1-P. By examining phosphoryl transfer from alpha 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 alpha Glc 1-P mainly for phosphoryl transfer to water. By applying D-fructose in 10-fold molar excess over alpha 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 alpha Glc 1-P.
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