Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in human ABO(H) blood group A and B glycosyltransferases

Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in... Abstract Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211, and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other CAZy family 6 glycosyltransferases, but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering. glycosyltransferases, human ABO(H) blood group enzymes, X-ray crystallography © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in human ABO(H) blood group A and B glycosyltransferases

Loading next page...
 
/lp/ou_press/conserved-residues-arg188-and-asp302-are-critical-for-active-site-135lhC0eVt
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
ISSN
0959-6658
eISSN
1460-2423
D.O.I.
10.1093/glycob/cwy051
Publisher site
See Article on Publisher Site

Abstract

Abstract Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211, and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other CAZy family 6 glycosyltransferases, but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering. glycosyltransferases, human ABO(H) blood group enzymes, X-ray crystallography © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

GlycobiologyOxford University Press

Published: Jun 5, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off