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Insights into substrate binding and utilization by hyaluronan synthase.

Zachery Stephens¹, Julia Karasinska^1,2, Jochen Zimmer^1,3

¹University of Virginia School of Medicine, 480 Ray C. Hunt Dr., Charlottesville, VA 22903.

Biorxiv : the Preprint Server for Biology

|November 24, 2025

Summary

This summary is machine-generated.

Hyaluronan synthase (HAS) uses a two-step process to bind UDP-glucuronic acid (UDP-GlcA), enhancing selectivity. This study reveals how HAS synthesizes hyaluronan, a crucial extracellular matrix component, with semi-selective sugar transfer.

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Area of Science:

Biochemistry
Structural Biology
Glycobiology

Background:

Hyaluronan (HA) is a vital vertebrate extracellular matrix polysaccharide.
HA functions in adhesion, lubrication, signaling, and spatial filling, essential for embryogenesis.
Hyaluronan synthase (HAS) synthesizes HA by polymerizing UDP-activated sugars and secreting the chain across the plasma membrane.

Purpose of the Study:

To elucidate the mechanism by which HAS recognizes and utilizes UDP-glucuronic acid (UDP-GlcA).
To understand how HAS couples substrate binding, catalysis, and product secretion.
To investigate the substrate specificity and potential promiscuity of HAS.

Main Methods:

Single-particle cryo-electron microscopy (cryo-EM) to determine HAS structures.
Biochemical assays to assess enzyme activity and substrate requirements.
Glycobiology techniques to analyze sugar transfer mechanisms.

Main Results:

A two-step substrate binding mechanism for UDP-GlcA was revealed, involving a proofreading pose prior to catalytic pocket insertion.
Specific basic residues in the active site were identified as crucial for sensing the UDP-GlcA carboxyl group.
HAS requires an acceptor sugar for UDP-GlcA turnover, confirming the necessity of a priming N-acetylglucosamine.
Cryo-EM revealed dodecylmaltoside in the active site, indicating HAS can transfer GlcA to non-canonical acceptors.

Conclusions:

HAS employs a proofreading mechanism to enhance UDP-GlcA selectivity.
The enzyme's activity is dependent on an acceptor substrate for initiating glycosyl transfer.
HAS exhibits semi-selectivity, capable of transferring glucuronic acid to non-canonical acceptors, offering insights into its biological function and potential engineering.