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Integrated Use of Biochemical, Native Mass Spectrometry, Computational, and Genome-Editing Methods to Elucidate the Mechanism of a Salmonella deglycase.

Abstract：

: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.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Sengupta A,Wu J,Seffernick JT,Sabag-Daigle A,Thomsen N,Chen TH,Capua AD,Bell CE,Ahmer BMM,Lindert S,Wysocki VH,Gopalan V

doi

10.1016/j.jmb.2019.08.017

subject

Has Abstract

pub_date

2019-11-08 00:00:00

pages

4497-4513

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(19)30541-8

journal_volume

431

pub_type

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Integrated Use of Biochemical, Native Mass Spectrometry, Computational, and Genome-Editing Methods to Elucidate the Mechanism of a Salmonella deglycase.