%0 Journal Article
%@ 1460-2423
%A Choudhary, Pravinkumar
%A Badmalia, Maulik D
%A Ashish, G
%A Rao, Alka
%D 2021
%F open:2678
%I Oxford University Press
%J Glycobiology
%K bacterial glycoproteins, O-GlcNActransferase, O-GlcNAcylation/flagella, protein glycosylation
%N 3
%P 275-287
%T Shape-function insights into bifunctional O-GlcNActransferase of Listeria monocytogenes EGD-e
%U http://crdd.osdd.net/open/2678/
%V 31
%X O-GlcNAcylation is an important post-translational modification of proteins. O-GlcNAcylated proteins have crucial roles in several cellular contexts both in eukaryotes and bacteria. O-GlcNActransferase (OGT) is the enzyme instrumental in O-GlcNAcylation of proteins. OGT is conserved across eukaryotes. The first bacterial OGT discovered is GmaR in Listeria monocytogenes. GmaR is a GT-2 family bifunctional protein that catalyzes glycosylation of the flagellin protein FlaA and controls transcription of flagellar motility genes in a temperature-dependent manner. Here, we provide methods for heterologous expression and purification of recombinant GmaR and FlaA, in vivo/in vitro glycosylation assays, analysis of the molecular form of recombinant GmaR and detailed enzyme kinetics. We study the structure and functional dynamics of GmaR. Using solution small-angle X-ray scattering and molecular modeling, we show that GmaR adopts an extended shape with two distinctly spaced structural units in the presence of cofactor Mg2+ and with donor UDP-GlcNAc and cofactor combined. Comparisons of restored structures revealed that in-solution binding of Mg2+ ions brings about shape rearrangements and induces structural-rigidity in hypervariable regions at the N-terminus of GmaR protein. Taking function and shape data together, we describe that Mg2+ binding enables GmaR to adopt a shape that can bind the substrate. The manuscript provides the first 3D solution structure of a bacterial OGT of GT-2 family and detailed biochemical characterization of GmaR to facilitate its future applications.
%Z The copyright of this article belongs to Oxford University Press