An enzyme-coupled continuous spectrophotometric assay for glycogen synthases

Abstract

The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.

Biochemical characterization of a novel bifunctional glycosyl-1-phosphate transferase involved in the exopolysaccharide biosynthesis

“…However, its application is limited due to the restricted availability of many substrates of radiochemicals. Recently, Wayllace reported that the continuous spectrophotometric assay for GTFs was similar to those values obtained with the radiochemical [18]. NMPK in reaction system not only can determine Ste5 transfer phospho glycosyl or glycosyl to acceptor, but it can also increase the sensitivity of the assay by twofold [25].…”

“…With a continuous coupled spectrophotometric assay for glycosyl-1-phosphate transferase [18,19], different NDP-sugar was used as glycosyl donor substrate, cytoplasmic membranes from different glycosyltransferase gene mutants as glycosyl-1-phosphate acceptor, the activity of Ste5 was measured in the presence of Nucleoside monophosphate kinase (NMPK), pyruvate kinase (PK) and lactate dehydrogenase (LDH). The NMPK transfers one phosphate group from ATP to NMP.…”

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis

“…Tre-6P Sase. Synthesis of Tre-6P from NDP-Glc and Glc-6P was assayed by measuring NADH formation at 340 nm via the coupled spectrophotometric method previously utilized for other glycosyl transferases [40,49,50] . The standard media contained 50 mM MOPS, pH 8.0, 5 mM MgCl 2 , 5 mM MnCl 2 , 0.3 mM phospho enol pyruvate, 0.3 mM NADH, 2.5 mM NDP-Glc, 5 mM Glc-6P, 2 U pyruvate kinase, 2 U lactate dehydrogenase and 0.2 mg/ml BSA and appropriately diluted enzyme in a final volume of 100 μl.…”

“…Assays were started by adding 20 μl of GSase dissolved in 20 mM triethanolamine-HCl, pH 8.0. GSase activity was alternatively measured with the same procedure used for Tre-6P Sase, but replacing Glc-6P by 2.5 mg/ml rabbit liver glycogen, according to [50] . The conversion of substrates to the expected products was confirmed using proton NMR spectroscopy.…”

Starch metabolism in green algae

“…This gene is phylogenetically related to glucan synthase in Chlamydiae, Cyanobacteria, and some Proteobacteria, possibly playing a role in linking the metabolism of the host and the endosymbiont. Because SSIII and SSIV enzymes uses preferentially ADPGlc in bacteria and plants , it is possible to postulate that C. paradoxa or, alternatively, the common ancestor of Viridiplantae and Glaucophytes may have used both, ADPGlc or UDPGlc for starch synthesis .…”