Mechanism of a cytosolic O -glycosyltransferase essential for the synthesis of a bacterial adhesion protein

Abstract

O-glycosylation of Ser and Thr residues is an important process in all organisms, which is only poorly understood. Such modification is required for the export and function of adhesin proteins that mediate the attachment of pathogenic Gram-positive bacteria to host cells. Here, we have analyzed the mechanism by which the cytosolic O-glycosyltransferase GtfA/B of Streptococcus gordonii modifies the Ser/Thr-rich repeats of adhesin. The enzyme is a tetramer containing two molecules each of GtfA and GtfB. The two subunits have the same fold, but only GtfA contains an active site, whereas GtfB provides the primary binding site for adhesin. During a first phase of glycosylation, the conformation of GtfB is restrained by GtfA to bind substrate with unmodified Ser/Thr residues. In a slow second phase, GtfB recognizes residues that are already modified with N-acetylglucosamine, likely by converting into a relaxed conformation in which one interface with GtfA is broken. These results explain how the glycosyltransferase modifies a progressively changing substrate molecule.

Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance

“…TarM in addition shows close structural resemblance to Streptococcus pneumoniae GtfA, a GTB O-GlcNAc transferase that is involved in the glycosylation of serine rich repeat adhesion proteins [66]. The DUF1975 domain of GtfA has been crystallographically observed to interact with a similar extended β-sheet domain of its GtfB co-activator, forming a hetero-tetramer [67]. As such, the vast difference in the derivation of the TarS and TarM trimerization domains supports the notion of separate evolutionary lineages and, along with other differences, elicits curiosity regarding the respective biological roles of these distant yet seemingly redundant enzymes.…”

Three glycosylated serine‐rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius

“…To examine whether GtfE/F function in the same manner as their GtfA/B homologues from S. agalactiae and S. gordonii by catalyzing the first step of SRR protein glycosylation through the incorporation of GlcNAc residues (Takamatsu et al ., ; Mistou et al ., ; Chaze et al ., ; Chen et al ., ), we analysed the supernatants of cells expressing SRR variants by Western blotting with succinylated wheat germ agglutinin (sWGA), a lectin specific for GlcNAc (Fig. E).…”

“…Glycosylation of SRR proteins is a sequential process in which GtfA/B are essential components. GtfA (also termed Gtf1) initiates the first step of the glycosylation process by transferring an N-acetylglucosamine (GlcNAc) residue to SRR proteins, whereas GtfB (also termed Gtf2) appears to act as a chaperone for GtfA for the glycosylation of its acceptor protein (Bu et al, 2008;Wu et al, 2010;Wu and Wu, 2011;Chen et al, 2016). This first step is crucial as loss of GtfA/B prevents SRR protein glycosylation (Li et al, 2014) and related protein function (Stephenson et al, 2002;Peng et al, 2008;Zhou and Wu, 2009).…”

Carb loading takes proteins on a ride

“…These SSR glycoproteins use a dedicated export system, the accessory secretion (Sec) system (6), which contains two homologues of the general Sec pathway (SecA2 and SecY2). The operon containing SecA2 and SecY2 also includes glycosyltransferases and other essential proteins (Asp1 to Asp5) that do not share homology to any protein of known function (7). It is known that these glycosyltransferases, named Nss and Gly in the pathogenic bacterium Streptococcus gordonii, are important for modification of the SRR adhesin GspB and that they act sequentially also in two other Streptococcus species after the general cytosolic O-glycosyltransferase GtfA/B modifies the adhesin's Ser/Thr-rich repeats (7).…”

“…The operon containing SecA2 and SecY2 also includes glycosyltransferases and other essential proteins (Asp1 to Asp5) that do not share homology to any protein of known function (7). It is known that these glycosyltransferases, named Nss and Gly in the pathogenic bacterium Streptococcus gordonii, are important for modification of the SRR adhesin GspB and that they act sequentially also in two other Streptococcus species after the general cytosolic O-glycosyltransferase GtfA/B modifies the adhesin's Ser/Thr-rich repeats (7). It is also known that Asp2 is a bifunctional protein required for the transport of the GspB and for its posttranslational modification (8) and that Asp2 has a putative catalytic region possessing a Ser-Asp-His catalytic triad.…”