(246e) In Vitro Enzymatic Synthesis of Hyaluronic Acid

Hyaluronic acid (HA) is a typical sequence-defined linear polysaccharide, consisting of alternating β-1,4-D-glucuronic acid (GlcUA) and β-1,3-N-acetyl-D-glucosamine (GlcNAc), with a high application potential in medicine, cosmetics, food industry, etc. Class I hyaluronan synthase (HAS) is responsible for the synthesis of HA in most creatures. The HAS catalysis is conducted in a processive manner, where HA is elongated by the intracellular GT domain utilizing UDP-GlcUA or UDP-GlcNAc as substrates, and subsequently translocated one monomer unit outwards through its transmembrane (TM) tunnel. Of particular interest is that HAS has a sole GT domain but undertakes the ordered connection of two substrates, which represents an extremely accurate control of substrate specificity. The in vitro reproduction of such an mechanism is interesting not only for expanding HA production routes but also on enriching our understanding of enzymology of HAS.

In this work, proteoliposome (PL) containing Streptococcus equisimilis HAS (SeHAS) was prepared and demonstrated for in vitro synthesis of HA. The alternating structure was maintained under high substrate concentration and unbalanced substrate ratio, confirmed by ¹H NMR and ESI-MS. Molecular docking, tunnel analysis and allosteric pathway identification detailed the molecular mechanism, as validated by site-directed mutagenesis. These molecular and process insights are helpful for the de novo design of enzymes for the synthesis of HA and other sequence-defined biopolymers in vitro.