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Title: Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily

Abstract

Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, the dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that doesmore » not require the typically conserved tyrosine residue.« less

Authors:
 [1];  [1];  [2];  [2];  [2];  [3]; ORCiD logo [1]
  1. Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, United States
  2. National Research Council Canada, Human Health Therapeutics, Ottawa, Ontario K1A 0R6, Canada
  3. Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, United States
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1409106
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 56; Journal Issue: 45
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Riegert, Alexander S., Thoden, James B., Schoenhofen, Ian C., Watson, David C., Young, N. Martin, Tipton, Peter A., and Holden, Hazel M. Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily. United States: N. p., 2017. Web. doi:10.1021/acs.biochem.7b00910.
Riegert, Alexander S., Thoden, James B., Schoenhofen, Ian C., Watson, David C., Young, N. Martin, Tipton, Peter A., & Holden, Hazel M. Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily. United States. doi:10.1021/acs.biochem.7b00910.
Riegert, Alexander S., Thoden, James B., Schoenhofen, Ian C., Watson, David C., Young, N. Martin, Tipton, Peter A., and Holden, Hazel M. Fri . "Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily". United States. doi:10.1021/acs.biochem.7b00910.
@article{osti_1409106,
title = {Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily},
author = {Riegert, Alexander S. and Thoden, James B. and Schoenhofen, Ian C. and Watson, David C. and Young, N. Martin and Tipton, Peter A. and Holden, Hazel M.},
abstractNote = {Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, the dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.},
doi = {10.1021/acs.biochem.7b00910},
journal = {Biochemistry},
number = 45,
volume = 56,
place = {United States},
year = {Fri Nov 03 00:00:00 EDT 2017},
month = {Fri Nov 03 00:00:00 EDT 2017}
}