Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeastderived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP- Hyaluronan synthases (HAS(s))1 are glycosyltransferases that catalyze the biosynthesis of hyaluronan (HA), a glycosaminoglycan generated at the plasma membrane of bacterial and eukaryotic cells. HA is an important structural component of the extracellular matrix and is also involved in a wide variety of biological process, such as tissue morphogenesis, cancer metastasis, wound healing, inflammation, and angiogenesis (1-5). This extended linear polymer is synthesized, apparently without any primer requirement, by alternate addition of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) units from the corresponding UDP-sugar donors (UDP-GlcUA and UDP-GlcNAc). HASs have been tentatively classified based on primary structure, molecular size, predicted membrane topology, and enzymological characteristics (6). Class I thus includes the streptococcal, the vertebrate (3 isoforms, HAS1-3), and the viral enzymes, whereas Class II contains a single member, the Pasteurella multocida enzyme. However, without knowledge of the three-dimensional structures or the catalytic mechanism, the two-class nomenclature system remains an hypothesis.Although biochemical properties and in vivo functional aspects of HAS proteins have been extensively studied, the mechanism of HA biosynthesis has remained unclear. The biosynthesis of chondroitin and heparan sulfates, and glycosaminoglycans with backbones of alternating hexuronic acid and hexosamine units, clearly occurs by a stepwise addition of monosaccharide units at the non-reducing termini of nascent chains (7), and the same mechanism of elongation has been demonstrated for the Escherichia coli K4 and K5 capsular polysaccharides (8 -9), as well as for chitin (10) and cellulose (11). In an early assessment of the directionality of HA chain elongation, Stoolmiller and Dorfman (12) used a particulate enzyme preparation from Group A Streptococcus (now known as HasA or spHAS). They identified products released by enzymatic digestion of metabolically labeled HA chains and concluded that chain elongation occurs at the nonreducing end, as in biosynthesis of other glycosaminoglycans (12). Prehm (13) approached the problem by digesting pulsechase-labeled HA produced by a teratocarcinoma cell membrane fraction and proposed, by contrast, that chain elongation occurs at the reducing end. This notion has by and large prevailed (se...