Abstract
Mycobacterium tuberculosis produces glycogen (also known as α-glucan) to help evade human immunity. This pathogen uses the GlgE pathway to generate glycogen rather than the more well known glycogen synthase GlgA pathway, which is absent in this bacterium. Thus, the building block for this glucose polymer is α-maltose-1-phosphate rather than an NDP-glucose donor. One of the routes to α-maltose-1-phosphate is now known to involve the GlgA homologue GlgM, which uses ADP-glucose as a donor and α-glucose-1-phosphate as an acceptor. To help compare GlgA (a GT5 family member) with GlgM enzymes (GT4 family members), the X-ray crystal structure of GlgM from Mycobacterium smegmatis was solved to 1.9 Å resolution. While the enzymes shared a GT-B fold and several residues responsible for binding the donor substrate, they differed in some secondary-structural details, particularly in the N-terminal domain, which would be expected to be largely responsible for their different acceptor-substrate specificities.
Highlights
Glycogen is a ubiquitous carbon-storage molecule composed of a glucose polymer constructed with -1,4 linkages and -1,6 branch points (Preiss, 2009)
GlgM was produced in E. coli BL21 Star cells (AMS Biotechnology Europe) which were grown at 18C to an OD600 of 0.6 in lysogeny broth (LB), when expression was induced with 0.5 mM isopropyl -d-1-thiogalactopyranoside (IPTG)
We switched our focus to the GlgM homologue from M. smegmatis, which shares 77% identity with the enzyme from M. tuberculosis
Summary
Glycogen is a ubiquitous carbon-storage molecule composed of a glucose polymer constructed with -1,4 linkages and -1,6 branch points (Preiss, 2009). The bacterial enzyme uses ADP-glucose as the sugar donor [GlgA, GT5 family (Lombard et al, 2014), EC 2.4.1.21, ADP- -d-glucose:(1!4) -d-glucan 4- -d-glucosyltransferase; Fig. 1a], while the eukaryotic enzyme uses UDP-glucose (GT3 family, EC 2.4.1.11). Another bacterial polymerase has been identified that does not utilize an NDP-glucose donor. All three polymerases use a GlgB branching enzyme to generate the mature branched polymer in conjunction with the appropriate polymerase. It is the combination of the polymerase and the branching enzyme that dictates the exact properties of the mature polymer (Rashid et al, 2016). The polymer generated by Mycobacterium tuberculosis (often referred to by the generic term -glucan) has been shown to be important in pathogenesis through its interaction with immune receptors (Koliwer-Brandl et al, 2016; Kalscheuer et al, 2019)
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