Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase

Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

“…To evaluate the role of the carboxylate side chain of Asp-372, we replaced this residue by Ser using site-directed mutagenesis. Using both a coupled spectrophotometric assay (29) and a more sensitive radiochemical assay (9), the D372S mutant showed no detectable activity, even following an overnight incubation (Table 2). In comparison, replacing the nearby Asp-371 residue (gray sphere), as well as the coordinating side chains.…”

“…Activity measurements using acceptor 2 were also attempted, but the lower level of activity of this substrate compared with acceptor 1 prevented the determination of K m and k cat parameters for any of the mutant enzymes. The lower level of activity with acceptor 2, and other compounds that first act as substrates for the ␣-(135)-transferase activity of GlfT2, has previously been reported (9,14,15,20,29) and is a topic of current study. ride substrates (Fig.…”

“…Mutagenesis was performed using the QuikChange II XL kit (Stratagene) with the oligonucleotide primer sequences given in supplemental Table 1. GlfT2 mutants were expressed and purified as described previously (29). Enzyme activity was measured using a coupled spectrophotometric assay (29) and a radiochemical assay (9) as described previously.…”

A tethering mechanism for length control in a processive carbohydrate polymerization

“…Because GlfT2 produces UDP after addition of each Galf residue to an acceptor, GlfT2 turnover was monitored with a continuous assay in which UDP production was coupled to NADH oxidation (34)(35)(36). Both compounds 2 and 5 were similarly competent for catalysis, with steady-state k cat values of 1.04 Ϯ 0.03 s Ϫ1 (compound 5) and 0.8 Ϯ 0.1 s Ϫ1 (compound 2), but the GlfT2-catalyzed reaction of compound 5 reached half-maximal steady-state velocity at a 27-fold lower concentration of compound 5 (K 1/2 value of 66 Ϯ 2 M) than with compound 2 (K 1/2 value of 1,800 Ϯ 700 M; Fig.…”

Carbasugar Analogues of Galactofuranosides: Pseudodisaccharide Mimics of Fragments of Mycobacterial Arabino­galactan

“…[26] The behaviour of the (1Ǟ5)-and (1Ǟ6)-linked pseudodisaccharides 19 and 18 as acceptor substrates and as inhibitors of GlfT2 was tested. [27] Neither pseudodisaccharide was recognised as a substrate. Although the enzyme has been reported to glycosylate disaccharide acceptors, trisaccharides are better substrates.…”