Abstract
Hyaluronic acid (HA) based biomaterials have several biomedical applications. HA biosynthesis is catalysed by hyaluronan synthase (HAS). The unavailability of 3-D structure of HAS and gaps in molecular understanding of HA biosynthesis process pose challenges in rational engineering of HAS to control HA molecular weight and titer. Using in-silico approaches integrated with mutation studies, we define a dictionary of sub-structural elements (SSE) of the Class I Streptococcal HAS (SeHAS) to guide rational engineering. Our study identifies 9 SSE in HAS and elucidates their role in substrate and polymer binding and polymer biosynthesis. Molecular modelling and docking assessment indicate a single binding site for two UDP-substrates implying conformationally-driven alternating substrate specificities for this class of enzymes. This is the first report hypothesizing the involvement of sites from SSE5 in polymer binding. Mutation at these sites influence HA production, indicating a tight coupling of polymer binding and synthase functions. Mutation studies show dispensable role of Lys-139 in substrate binding and a key role of Gln-248 and Thr-283 in HA biosynthesis. Based on the functional architecture in SeHAS, we propose a plausible three-step polymer extension model from its reducing end. Together, these results open new avenues for rational engineering of Class I HAS to study and regulate its functional properties and enhanced understanding of glycosyltransferases and processive enzymes.
Highlights
Hyaluronic acid (HA) is a hetero-biopolymer of repeating β-1,4-D-glucuronic acid and β-1,3-N-acetyl-Dglucosamine units[1]
The model was obtained with cellulose synthase template (PDB: 4P00).The template enzyme shares a high functional similarity and a low sequence similarity (~15%) with SeHAS25
The present study provides a dictionary of substructural elements of functional importance that could act as framework for rational engineering of hyaluronan synthase (HAS) class I family of enzymes
Summary
Hyaluronic acid (HA) is a hetero-biopolymer of repeating β-1,4-D-glucuronic acid and β-1,3-N-acetyl-Dglucosamine units[1]. Random and directed mutation studies have been conducted in Streptococcal and mammalian HAS systems to identify property-influencing sites These include point mutations in membrane region[18], cysteine residues[19], select conserved residues[20], upstream and downstream regions of conserved residues[21], and C-terminal end of the enzyme[22,23]. Other characterized glycosyltransferases including cellulose synthase polymerize at the non-reducing end[24] The implications of these differences on 3-D structure, active site architecture, substrate binding sites and mechanism is completely unknown. The objective of this study is to characterize the functional components of Class I HAS to enhance molecular understanding of polymer biosynthesis and provide a framework for rational engineering of HAS. We considered Streptococcal HAS (SeHAS), a biochemically characterized HAS as the representative member for analysis[9]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
