Integrated Use of Biochemical, Native Mass Spectrometry, Computational, and Genome-Editing Methods to Elucidate the Mechanism of a Salmonella deglycase.
Amadori products
Salmonella
deglycase
enzyme mechanism
Journal
Journal of molecular biology
ISSN: 1089-8638
Titre abrégé: J Mol Biol
Pays: Netherlands
ID NLM: 2985088R
Informations de publication
Date de publication:
08 11 2019
08 11 2019
Historique:
received:
01
04
2019
revised:
27
08
2019
accepted:
28
08
2019
pubmed:
8
9
2019
medline:
17
6
2020
entrez:
8
9
2019
Statut:
ppublish
Résumé
Salmonellais a foodborne pathogen that causes annually millions of cases of salmonellosis globally, yet Salmonella-specific antibacterials are not available. During inflammation, Salmonella utilizes the Amadori compound fructose-asparagine (F-Asn) as a nutrient through the successive action of three enzymes, including the terminal FraB deglycase. Salmonella mutants lacking FraB are highly attenuated in mouse models of inflammation due to the toxic build-up of the substrate 6-phosphofructose-aspartate (6-P-F-Asp). This toxicity makes Salmonella FraB an appealing drug target, but there is currently little experimental information about its catalytic mechanism. Therefore, we sought to test our postulated mechanism for the FraB-catalyzed deglycation of 6-P-F-Asp (via an enaminol intermediate) to glucose-6-phosphate and aspartate. A FraB homodimer model generated by RosettaCM was used to build substrate-docked structures that, coupled with sequence alignment of FraB homologs, helped map a putative active site. Five candidate active-site residues-including three expected to participate in substrate binding-were mutated individually and characterized. Native mass spectrometry and ion mobility were used to assess collision cross sections and confirm that the quaternary structure of the mutants mirrored the wild type, and that there are two active sites/homodimer. Our biochemical studies revealed that FraB Glu214Ala, Glu214Asp, and His230Ala were inactive in vitro, consistent with deprotonated-Glu214 and protonated-His230 serving as a general base and a general acid, respectively. Glu214Ala or His230Ala introduced into the Salmonella chromosome by CRISPR/Cas9-mediated genome editing abolished growth on F-Asn. Results from our computational and experimental approaches shed light on the catalytic mechanism of Salmonella FraB and of phosphosugar deglycases in general.
Identifiants
pubmed: 31493410
pii: S0022-2836(19)30541-8
doi: 10.1016/j.jmb.2019.08.017
pmc: PMC6885551
mid: NIHMS1539371
pii:
doi:
Substances chimiques
Bacterial Proteins
0
Hydrolases
EC 3.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
4497-4513Subventions
Organisme : NIAID NIH HHS
ID : R01 AI116119
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI140541
Pays : United States
Informations de copyright
Copyright © 2019 Elsevier Ltd. All rights reserved.
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