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Shape-function insights into bifunctional O-GlcNActransferase of Listeria monocytogenes EGD-e

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Shape-function insights into bifunctional O-GlcNActransferase of Listeria monocytogenes EGD-e
 
Creator Choudhary, Pravinkumar
Badmalia, Maulik D.
Ashish, Ganguly
Rao, Alka
 
Subject QR Microbiology
 
Description 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 hyper-variable 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.
 
Publisher OXFORD ACADEMIC
 
Date 2021-03-03
 
Type Article
PeerReviewed
 
Relation https://academic.oup.com/glycob/article/31/3/275/5889979
http://crdd.osdd.net/open/2745/
 
Identifier Choudhary, Pravinkumar and Badmalia, Maulik D. and Ashish, Ganguly and Rao, Alka (2021) Shape-function insights into bifunctional O-GlcNActransferase of Listeria monocytogenes EGD-e. GLYCOBIOLOGY, 31 (3). pp. 275-287.