Insights into the structure and function of membrane-integrated processive glycosyltransferases

Abstract

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).

Figure 1

Membrane-integrated processive GTs synthesize and secrete diverse polysaccharides. The synthases may be part of multi-component complexes or function on their own. The catalytically active subunits (colored brown) share an intracellular GT and a membrane-integrated domain. Alginate consists of mannuronic (yellow) and guluronic acid (green), cellulose of glucose (beige), PNAG of NAG (gray), chitin of NAG, and HA of NAG and GA (magenta) units. A dashed circle indicates the binding site for the signaling molecule cyclic-di-GMP. Lower panel: The enzymes catalyze the transfer of a nucleotide diphosphate (NDP)-activated sugar (black hexagon) to another glycosyl unit, thereby generate NDP as a second reaction product. Among the synthases shown, HAS is the only enzyme that appears to elongate the polymer at its reducing end, thereby generating an UDP-attached polysaccharide. OM, IM: Outer and inner membrane.

Figure 2

Sequence alignment and predicted secondary structure of selected family-2 GTs. (a) Predicted TM topology of Rhodobacter sphaeroides BcsA, Homo sapiens HAS2, Pseudomonas aeruginosa Alg8, Klebsiella pneumoniae PgaC, and Saccharomyces cerevisiae CHS3. Topology diagrams are shown from the N to the C terminus, labeled N and C for BcsA. GT: glycosyltransferase domain. The membrane region is shown as a blue rectangle. Topologies were predicted with TOPCONS [71]. (b) Multiple sequence alignment of the sequences used in (a). The sequences were aligned in CLUSTALW [72] and predicted TM topologies and secondary structures were used to manually refine the alignment. Predicted TM helices and secondary structure elements are shown as gray bars (TM helices) and green columns and yellow arrows for α-helices and β-strands, respectively. Conserved sequence motifs are framed with a red box and numbered 1–4.

Figure 3

Chemical diversity of polysaccharides. Coordinates for the shown oligosaccharides were obtained and adjusted from pdb entries 4P02 (cellulose), 3AFL (alginate), 2JCQ (HA), 4P7R (PNAG), and 3WH1 (chitin). The carbon atoms of the individual sugars are colored as in Figure 1, oxygen and nitrogen atoms are colored red and blue, respectively. The polymer’s reducing ends are labeled with an asterisk.

Figure 4

Pore organization of bacterial cellulose synthase subunit BcsA. BcsA’s TMHs are shown as a gray surface with the exception of TMH5 and IF2, which contains the ‘QxxRW’ motif. The GT domain is shown as a blue cartoon and cellulose and UDP are shown as sticks with blue and yellow carbon atoms, respectively. Selected residues belonging to the conserved DDG (D180), DxD (D246 and 248), TED (D343), and Q/ LxxRW (W383) motifs are shown as sticks. Cellulose synthase elongates the polymer at the non-reducing end, which is stabilized at the acceptor site within the catalytic pocket.