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:
Lipopolysaccharide glycosyl transferase, WbbF
Bacteria
WbbF of Salmonella enterica serovar Borreza plasmid pWQ799 (Q52257)
The hyaluronan (hyaluronate) synthase1/exporter, HAS1 of 578 aas and 6 or 7 TMSs in a 1 or 2 TMS(s) (N-terminal) + 5 TMSs (C-terminal) arrangement. Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronate residues that is ubiquitously expressed in the vertebrate extracellular matrix. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis (Maloney et al. 2022). Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors. Maloney et al. 2022 described five cryo-EM structures of a viral HAS homologue in different states during substrate binding and initiation of polymer synthesis. HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. A detailed model for the formation of an acidic extracellular heteropolysaccharide is proposed that provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body (Maloney et al. 2022).
Animals
HAS1 of Homo sapiens (Q92839)
Uncharacterized protein, YaiP, of 398 aas with 1 N-terminal TMS and 3 C-terminal TMSs.
YaiP of E. coli
Chitin synthase 3, Chs3, of 1165 aas and 7 TMSs in a 2 + 1 + 4 TMS arrangement. Chitin biosynthesis in yeast is accomplished by three chitin synthases (Chs) termed Chs1 (TC# 4.D.1.1.17), Chs2 (TC# 4.D.1.1.16) and Chs3, the last which accounts for most of the chitin deposited within the cell wall. While the overall structures of Chs1 and Chs2 are similar to those of other chitin synthases from fungi and arthropods, Chs3 lacks some of the C-terminal transmembrane helices raising questions regarding its structure and topology. Gohlke et al. 2017 determined aspects of the catalytic domain, the chitin-translocating channel and the interfacial helices in between. They identified an amphipathic, crescent-shaped alpha-helix attached to the inner side of the membrane that may control the channel entrance and a finger helix pushing the polymer into the channel. Chitin synthases form oligomeric complexes, which may be necessary for the formation of chitin nanofibrils. They detected oligomeric complexes at the bud neck, the lateral plasma membrane, and in membranes of Golgi vesicles (Gohlke et al. 2017). The combined action of two independent but redundant endocytic recycling mechanisms, together with distinct labels for vacuolar degradation, determines the final fate of the intracellular traffic of the Chs3 protein, allowing yeast cells to regulate morphogenesis, depending on environmental constraints (Arcones et al. 2016).
Chs3 of Saccharomyces cerevisiae
Glycosyltransferase (Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase) of 322 aas with 2-3 C-terminal TMSs, ArnC. Catalyzes the transfer of 4-deoxy-4-formamido-L-arabinose from UDP to undecaprenyl phosphate. The modified arabinose is attached to lipid A and is required for resistance to polymyxin and cationic antimicrobial peptides (Breazeale et al. 2002). This protein does not catalyze transport and is homologous to other enzymes in the GT2 family only in the N-terminal cytoplasmic glycosyl transferase domain.
ArnC of E. coli
GtrB of 318 aas and 2 TMSs. The 3.0 Å resolution crystal structure of GtrB, a glucose-specific polyisoprenyl-glycosyltransferase (PI-GT) from Synechocystis, has been reported showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle (Ardiccioni et al. 2016). The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. A role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site has been proposed (Ardiccioni et al. 2016).
GtrB of Synechocystis sp.
Poly-beta-1,6-N-acetyl-D-glucosamine (NAG) synthase with a 5 TMS + hydrophilic glycosyl transferase domain +4 or 5 more TMSs. Only the middle hydrophilic glycosyltransferase domain is homologous to other members of the family.
Poly-β-1,6-NAG synthase of Candidatus Odinarchaeota archaeon
Chitin synthase II, CHS2, of 963 aas and 7 TMSs in a 5 + 2 TMS arrangement. It is essential for septum formation and cell division and is required for maintaining normal cell morphology.
CHS2 of Saccharomyces cerevisiae (Baker's yeast)
Chitin synthase I, CHS1 of 1131 aas and 7 TMSs in a 5 + 2 TMS arrangement. It is necessary for septum formation and repair, especially under certain adverse conditions.
CHS1 of Saccharomyces cerevisiae (Baker's yeast)
Hyaluronan synthase 2, HAS2, of 552 aas and 7 or 8 TMSs with 2 or 3 TMSs at the N-terminus and 5 TMSs at the C-terminus. It is 50% identical to HAS1 (TC# 4.D.1.1.10). It catalyzes the addition of GlcNAc or GlcUA monosaccharides to the nascent hyaluronan polymer and is essential to hyaluronan synthesis, a major component of most extracellular matrices. It plays a structural role in tissues architectures and regulates cell adhesion, migration and differentiation. Autophagic degradation of HAS2 in endothelial cells provides a mechanism for the regulation of angiogenesis (Chen et al. 2020). This is one of three isoenzymes responsible for cellular hyaluronan synthesis, and it is responsible for the synthesis of high molecular mass hyaluronan. It is produced in increased amounts in the mole rat where it extends life span and decreases the incidence of cancer. "Abundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent-the naked mole-rat (Zhang et al. 2023).
NAS2 of Homo sapiens
IcaA of 412 aas and 4 (1 at the N-terminus + 3 at the C-terminus) TMSs.
Bacteria
IcaA of Staphylococcus epidermidis (Q54066)
PgaC or YcdQ of 441 aas with a 5 (2 (N-terminal) + 3 (C-terminal)) TMS topology. TC Blast retrieves 4.D.3.1.5 and 4.D.2.1.9 with scores of e-13 and e-8, respectively, for the hydrophilic catalytic domains, demonstrating homology. This enzyme is an N-acetylglucosaminyltransferase that
catalyzes the polymerization of
UDP-N-acetylglucosamine to produce the linear homopolymer,
poly-beta-1,6-N-acetyl-D-glucosamine (PGA), a biofilm adhesin
polysaccharide (Cerca and Jefferson 2008). May function with PgaD, a 137 aa, 2 TMS protein (P69432). The polysaccharide is partially deacetylated by PgaB (P75906) in preparation for export across the outer membrane by PgaA (TC# 1.B.55.1.1).
Bacteria
PgaC of E. coli (P75905)
Hyaluronate synthase, HasA
Bacteria
HasA of Streptococcus pyogenes (P0C0H0)
The hyaluronan (hyaluronate) synthase/exporter, HAS
Bacteria
HAS of Streptococcus dysgalactiae subsp. equisimilis (O50201)
Uncharacterized protein of 354 aas and 4 TMSs.
Proteobacteria
UP of Beggiatoa sp
Alginate synthesis-related protein of 882 aas and 5 TMSs
Aquificae
Alginate synthesis-related protein of Aquifex aeolicus
Synthase of type 3 pneumoclccal capsular polysaccharide of 417 aas and 4 TMSs, Cap3B.
Firmicutes
Cap3B of Streptococcus pneumoniae
Glycosyl transferase involved in alginate biosynthesis of 494 aas and ~ 5 TMSs, Alg8 (Fata Moradali et al. 2015).
Proteobacteria
Alg8 of Pseudomonas aeruginosa
Putative glycosyl transferase of 543 aas and 5 TMSs in a 2 (N-terminal) + 3 (C-terminal) TMS arrangement
GTr of Candidatus Saccharibacteria bacterium
Putative glycosyl transferase of 585 aas and 5 TMSs in a 2 + 3 TMS arrangement
Glycosyltransferase of Brevibacterium casei
Putative glycosyl transferase of 557 aas and 5 TMSs in a 2 + 3 TMS arrangement
Conserved membrane protein of Candidatus Saccharibacteria
Glycosyl transferase_GTA with a C-terminal TRP domain of 411 aas.
Chlamydiae
GTA of Parachlamydia acanthamoebae
UDP glucose-lipopolysaccharide glycosyl transferase of 252 aas.
Proteobacteria
UDP glucose LPS glycosyl transferase of Haemophilus parasuis
Glycosyl transferase of 1,435 aas
Proteobacteria
Glycosyl transferase of Geobacter lovleyi
Glycosyl transferase of 1435aas, RfbC. Functions with ABC exporter, TC#3.A.1.103.3
Proteobacteria
RfbC of Myxococcus xanthus (Q50864)
Uncharacterized protein of 252 aas and 0 TMSs. Annotated as a family 2 glycosyltransferase.
UP of Candidatus Beckwithbacteria bacterium
UDP glucose:ceremide glucosyl transferase of 394 aas and 4 TMSs in a 1 + 3 TMS arrangment. May serve as a flippase (Ichikawa et al. 1996).
Animals
Ceramide glucosyl transferase of Homo sapiens
Ceramide glucosyl transferase, Cgt. of 479 aas
Fungi
Cgt of Ogataea parapolymorpha (Yeast) (Hansenula polymorpha)
Ceramide glucosyl transferase, Cgt, of 390 aas
Nitrospirae (Bacteria)
Cgt of Thermodesulfovibrio yellowstonii
Hopinoid biosynthesis associated glycosyl transferase of 390 aas and ~5 TMSs (Schmerk et al. 2015).
Armatimonadetes (Bacteria)
HpnI of Chthonomonas calidirosea