Structure/function analysis of the Pasteurella multocida heparosan synthases: towards defining enzyme specificity and engineering novel catalysts

Authors
Otto, N.J.
Masuko, S.
Mayer, A.
Tanner, M.E.
Linhardt, Robert J.
DeAngelis, P.L.
ORCID
https://orcid.org/0000-0003-2219-5833
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Issue Date
2012
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Terms of Use
Attribution 3.0 United States
CC BY : this license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. Credit must be given to the authors and the original work must be properly cited.
Full Citation
Structure/function analysis of the Pasteurella multocida heparosan synthases: towards defining enzyme specificity and engineering novel catalysts, N. J. Otto, S. Masuko, A. Mayer, M. E. Tanner, Robert J. Linhardt, P. L. DeAngelis, Journal of Biological Chemistry, 287, 7203–7212, 2012.
Abstract
The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.
Description
Journal of Biological Chemistry, 287, 7203–7212
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Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
Publisher
The American Society for Biochemistry and Molecular Biology (ASBMB) and Elsevier
Relationships
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
https://harc.rpi.edu/
Access
Open Access
CC BY — Creative Commons Attribution