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4.D.1 The 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. SERCA interacts with chitin synthase and participates in cuticular chitin biogenesis in Drosophila (Zhu et al. 2022). The structures, catalysis, chitin transport, and selective inhibition of chitin synthases have been reviewed (Chen et al. 2023).

Most fungi have multiple chitin synthases (CSs) that make chitin at different sites on the cell surface, at different times during growth, and in response to cell wall stress. The structure-based model for CS function is for transfer of GlcNAc from UDP-GlcNAc at the cytoplasmic face of the plasma membrane to the non-reducing end of a growing chitin chain, which is concomitantly translocated through a transmembrane channel formed by the synthase. CSs may 'self-prime' by hydrolyzing UDP-GlcNAc to yield GlcNAc, and A CS's active site is not continuously occupied by a nascent chitin chain; rather, CSs can release chitin chains, then re-initiate, and therefore synthesize chitin chains in bursts (Orlean and Funai 2019). 

Three distinct types of hyaluronan (HA) synthase (HAS) bifunctional glycosyltransferases (GTs) with disparate architectures and reaction modes are known (DeAngelis and Zimmer 2023). Class I membrane-integrated HASs employ a processive chain elongation mechanism and secrete HA across the plasma membrane. This complex operation is accomplished by functionally integrating a cytosolic catalytic domain with a channel-forming transmembrane region. Class I enzymes, containing a single GT family-2 (GT-2) module that adds both monosaccharide units to the nascent chain, are further subdivided into two groups that construct the polymer with opposite molecular directionalities: Class I-R and I-NR elongate the HA polysaccharide at either the reducing or the non-reducing end, respectively. In contrast, Class II HASs are membrane-associated peripheral synthases with a non-processive, non-reducing end elongation mechanism using two independent GT-2 modules (one for each type of monosaccharide) and require a separate secretion system for HA export. DeAngelis and Zimmer 2023 discussed recent mechanistic insights into HA biosynthesis that promise biotechnological benefits and exciting engineering approaches.

References associated with 4.D.1 family:

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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
Chen, C.G., M.A. Gubbiotti, A. Kapoor, X. Han, Y. Yu, R.J. Linhardt, and R.V. Iozzo. (2020). Autophagic degradation of HAS2 in endothelial cells: A novel mechanism to regulate angiogenesis. Matrix Biol 90: 1-19. 32084457
Chen, D.D., Z.B. Wang, L.X. Wang, P. Zhao, C.H. Yun, and L. Bai. (2023). Structure, catalysis, chitin transport, and selective inhibition of chitin synthase. Nat Commun 14: 4776. 37553334
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
DeAngelis, P.L. and J. Zimmer. (2023). Hyaluronan synthases; mechanisms, myths, & mysteries of three types of unique bifunctional glycosyltransferases. Glycobiology. [Epub: Ahead of Print] 37769351
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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
Maloney, F.P., J. Kuklewicz, R.A. Corey, Y. Bi, R. Ho, L. Mateusiak, E. Pardon, J. Steyaert, P.J. Stansfeld, and J. Zimmer. (2022). Structure, substrate recognition and initiation of hyaluronan synthase. Nature 604: 195-201. 35355017
Orlean, P. and D. Funai. (2019). Priming and elongation of chitin chains: Implications for chitin synthase mechanism. Cell Surf 5: 100017. 32743134
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
Zhang, Z., X. Tian, J.Y. Lu, K. Boit, J. Ablaeva, F.T. Zakusilo, S. Emmrich, D. Firsanov, E. Rydkina, S.A. Biashad, Q. Lu, A. Tyshkovskiy, V.N. Gladyshev, S. Horvath, A. Seluanov, and V. Gorbunova. (2023). Increased hyaluronan by naked mole-rat Has2 improves healthspan in mice. Nature. [Epub: Ahead of Print] 37612507
Zhu, W., Y. Duan, J. Chen, H. Merzendorfer, X. Zou, and Q. Yang. (2022). SERCA interacts with chitin synthase and participates in cuticular chitin biogenesis in Drosophila. Insect Biochem Mol Biol 145: 103783. 35525402