Integrated Use of Biochemical, Native Mass Spectrometry, Computational, and Genome-Editing Methods to Elucidate the Mechanism of a Salmonella deglycase
| Autor: | Charles E. Bell, Brian M. M. Ahmer, Nicholas Thomsen, Anice Sabag-Daigle, Steffen Lindert, Venkat Gopalan, Tien-Hao Chen, Anindita Sengupta, Jikang Wu, Vicki H. Wysocki, Justin T Seffernick, Angela Di Capua |
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| Rok vydání: | 2019 |
| Předmět: |
Salmonella
Hydrolases Mutant Sequence alignment medicine.disease_cause Mass Spectrometry Article Substrate Specificity 03 medical and health sciences 0302 clinical medicine Genome editing Bacterial Proteins Structural Biology medicine CRISPR Molecular Biology 030304 developmental biology chemistry.chemical_classification Gene Editing 0303 health sciences biology Wild type Active site Enzyme chemistry Biochemistry Mutation biology.protein 030217 neurology & neurosurgery |
| Zdroj: | J Mol Biol |
| ISSN: | 1089-8638 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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