Hyaluronan synthases; mechanisms, myths, & mysteries of three types of unique bifunctional glycosyltransferases

Abstract

Hyaluronan (HA), the essential [-3-GlcNAc-1-β-4-GlcA-1-β-]n matrix polysaccharide in vertebrates and molecular camouflage coating in select pathogens, is polymerized by "HA synthase" (HAS) enzymes. The first HAS identified three decades ago opened the window for new insights and biotechnological tools. This review discusses current understanding of HA biosynthesis, its biotechnological utility, and addresses some misconceptions in the literature. HASs are fascinating enzymes that polymerize two different UDP-activated sugars via different glycosidic linkages. Therefore, these catalysts were the first examples to break the "one enzyme/one sugar transferred" dogma. Three distinct types of these bifunctional glycosyltransferases (GTs) with disparate architectures and reaction modes are known. Based on biochemical and structural work, we present an updated classification system. 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. We discuss recent mechanistic insights into HA biosynthesis that promise biotechnological benefits and exciting engineering approaches.

Keywords: biosynthesis; catalysis; enzyme; polymerization; polysaccharide.

Fig. 1

Schematic of HA matrix, polymer structure and synthases. A) Eukaryotic cells have an HA polysaccharide coating (along with various proteins, not shown) on their extracellular surfaces (HA matrix and chain) while select microbial cells have an HA-rich capsule. B) HA polysaccharide has a [-3-GlcNAc-1-β-4-GlcA-1-β-]_n repeating structure where n can range up to 10⁴. C) Depending on the species, one of three types of HA synthase enzymes (see Table 1) polymerizes the monosaccharides from UDP-sugar donors into the HA chain that is then secreted or transported into the extracellular space. Class II HASs synthesize HA in vivo on a glycolipid anchor (indicated by a lipid-linked square) generated by enzymes encoded in the corresponding capsular polysaccharide biosynthesis gene operon.

Fig. 2

Schematic models of HA biosynthesis past and present. A) The first reported model of the formation of HA biosynthesis (adapted from Markovitz et al. 1959; reproduced with permission from J. Biol. Chem.) where alternating attack by incoming UDP-sugars results in the formation of sugar repeats. B) Modern models of the two types of Class I HAS; while both have channels to secrete the identical HA chain, the two types differ in molecular directionality of chain elongation. For I-R synthases, the monosaccharide of the UDP-donor attacks the reducing end of the nascent UDP-HA chain intermediate; these enzymes have been proposed to dimerize to simultaneously bind UDP-linked HA and donor sugars. On the other hand, for I-NR HASs, the terminal monosaccharide at the non-reducing end of the nascent HA chain attacks the UDP-sugar donor.

Fig. 3

Structural models of the various HASs. The experimentally determined structure of CvHAS (PDB: 7SPA; Class I-NR) is shown as a cartoon with TM helices colored in gray, interface helices in blue, and the catalytic GT-2 domain in magenta and cyan for beta-strands and helices, respectively. The priming GlcNAc monosaccharide is shown with orange spheres for carbon atoms. AlphaFold2 (AF) models are shown for HAS from Homo sapiens (Hs; Class I-NR), Streptococcus equisimilis (Se; Class I-R), and Pasteurella multocida (Pm; Class II). The putative HA translocation pathways are shown as a blue dashed line. SeHAS is likely to form an HA translocation channel at a dimer interface (the second HAS polypeptide is shown in light blue to the left 3D structure), but further work is needed to confirm. Aspartate residues (the first Asp in D-X-D motif) implicated in substrate binding are shown and labeled for all species. The indicated Trp (W) residues mark the acceptor position above the Class I catalytic pockets.

Fig. 4

Model of HA biosynthesis by Class I-NR enzymes. The postulated steps of polysaccharide initiation and production in order from left to right. HAS generates a monosaccharide primer by hydrolyzing a UDP-GlcNAc substrate. Substrate selectivity is determined by the nature of the accepting glycosyl unit. GlcNAc’s acetamido group prevents binding of a UDP-GlcNAc substrate and a GlcA acceptor is incompatible with binding of a UDP-GlcA substrate. The control of HA chain elongation and termination is not yet understood.