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4.D.1 The Putative Vectorial Glycosyl Polymerization (VGP) Family

Several processive glycosyl transferases have been implicated in transport. WbbF of Salmonella enterica sv. Borreze is one such enzyme. It consists of 459 amino acyl residues with 4 TMSs. It has a periplasmic domain immediately following a large cytoplasmic domain in which the glycosyl transferase activity resides. Cps35 of Streptococcus pneumoniae, HasA of Streptococcus pyogenes, IcaA of Staphylococcus epidermidis and several chitin synthases represent other examples of potential vectorial polymerases. The C-terminus of WbbF is thought to have pore-forming activity. Transport by these enzymes may occur by 'vectorial polymerization,' a group translocation process. Over 1000 homologues of WbbF are found in Gram-positive bacteria, Gram-negative bacteria, cyanobacteria, archaea and plants. These proteins vary in size from about 250 to about 750 amino acyl residues. However most of the functionally characterized bacterial glycosyl transferases are of 400-500 residues.

Hyaluronan (HA), an extracellular linear polysaccharide of alternating N-acetyl-glucosamine and glucuronic acid residues, is ubiquitously expressed in vertebrates, where it affects a broad spectrum of physiological processes, including cell adhesion, migration and differentiation. The HA polymer is synthesized on the cytosolic side of the cell membrane by the membrane-embedded hyaluronan synthase (HAS). The bacterial HAS from Streptococcus equisimilis (Se) shares a similar transmembrane topology and significant sequence identity with human HASs and likely synthesizes HA by the same mechanism. Hubbard et al. (2012) demonstrated that the Se-HAS is both necessary and sufficient to translocate HA in a reaction that is tightly coupled to HA elongation. The purified Se-HAS was reconstituted into proteoliposomes (PLs) where it synthesized and translocated HA. In vitro synthesized, high-molecular-weight HA remained tightly associated with the intact PLs in sedimentation experiments. Most importantly, the newly formed HA is protected from enzymatic degradation by hyaluronidase unless the PLs were solubilized with detergent, thereby demonstrating that HA was translocated into the lumen of the vesicle. HA synthesis and translocation are spatially coupled events. The coupled synthesis and membrane translocation of a biopolymer may be applicable to the synthesis of other biopolymers including chitin and cellulose.

Hyaluronan (HA) biosynthesis by HA synthase (HAS) has been studied for over six decades.  Class I family members include mammalian and streptococcal HASs, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains (Weigel 2015). HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, it is believed to create an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. A synthase pore-mediated polysaccharide translocation process may occur via the Pendulum mechanism This is an ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP compounds. The synthesis of chitin-UDP oligomers by HAS uses a reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These findings indicate that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers (Weigel 2015).

Although some polysaccharide biosynthetic substrates are moved across the membrane to sites of polysaccharide synthesis by separate transporter proteins before being incorporated into polymers by glycosyltransferase proteins, many polysaccharide biosynthetic enzymes appear to have both transporter and transferase activities. In these cases, the biosynthetic enzymes utilize substrate on one side of the membrane and deposit the polymer product on the other side. Davis (2012) has discussed structural characteristics of plant cell wall glycan synthases that couple synthesis with transport, drawing on what is known about such dual-function enzymes in other species.

References associated with 4.D.1 family:

Cerca, N. and K.K. Jefferson. (2008). Effect of growth conditions on poly-N-acetylglucosamine expression and biofilm formation in Escherichia coli. FEMS Microbiol. Lett. 283: 36-41. 18445167
Davis, J.K. (2012). Combining polysaccharide biosynthesis and transport in a single enzyme: dual-function cell wall glycan synthases. Front Plant Sci 3: 138. 22737159
Fata Moradali, M., I. Donati, I.M. Sims, S. Ghods, and B.H. Rehm. (2015). Alginate Polymerization and Modification Are Linked in Pseudomonas aeruginosa. MBio 6:. 25968647
Gohlke, S., S. Muthukrishnan, and H. Merzendorfer. (2017). In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae. Int J Mol Sci 18:. 28346351
Hubbard, C., J.T. McNamara, C. Azumaya, M.S. Patel, and J. Zimmer. (2012). The hyaluronan synthase catalyzes the synthesis and membrane translocation of hyaluronan. J. Mol. Biol. 418: 21-31. 22343360
Ichikawa, S., H. Sakiyama, G. Suzuki, K.I. Hidari, and Y. Hirabayashi. (1996). Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis. Proc. Natl. Acad. Sci. USA 93: 12654. 8901638
Schmerk, C.L., P.V. Welander, M.A. Hamad, K.L. Bain, M.A. Bernards, R.E. Summons, and M.A. Valvano. (2015). Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid-producing bacteria. Environ Microbiol 17: 735-750. 24888970
Weigel, P.H. (2015). Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior. Int J. Cell Biol. 2015: 367579. 26472958