Elucidating the unusual reaction kinetics of glucuronyl C5-epimerase

Vaidyanathan, Deepika
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Dordick, Jonathan S.
Linhardt, Robert J.
Koffas, Mattheos A. G.
Wang, Chunyu
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Chemical engineering
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Chemoenzymatic synthesis of heparin, a multienzyme process, represents an emerging alternative to animal sourced heparan sulfate and heparin. Glucuronyl C5-epimerase is the first enzyme that acts on the substrate polysaccharide, N-sulfoheparosan, chemically prepared from heparosan isolated from capsule of E. coli K5. Since the epimerization reaction is associated with no easily measured physical or chemical change, direct assays to determine the activity of C5-epimerase has been a challenge. As a consequence, understanding of its reaction mechanism and its application in heparin and heparan sulfate biosynthesis is quite difficult. The goal of this thesis is to elucidate the kinetics and reaction mechanism of C5-epimerase within the context of the chemoenzymatic scheme. Polysaccharide substrates directly relevant to the production of bioengineered heparin is used. Novel NMR-based online and offline assays were developed that leverages epimerase mediated hydrogen-deuterium exchange. Apparent kinetic parameters were determined for both the forward and pseudo-reverse reactions. Atypical sigmoidal behavior was observed for the forward reaction. We have also elucidated the specificity and action pattern of C5-epimerase using polysaccharide substrates. We demonstrate that C5-epimerase specificity relates directly to the location of the N-acetylglucosamine (GlcNAc) residues within the chain. Critical information gleamed from this research leads to better understanding the fundamental mechanism of C5-epimerase and ultimately enables the optimization of large-scale production of bioengineered heparin
August 2020
School of Engineering
Dept. of Chemical and Biological Engineering
Rensselaer Polytechnic Institute, Troy, NY
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