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Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine
Amadori rearrangement products are stable sugar‐amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose‐lysine (F‐Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley & Sons, Inc.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10285752/ https://www.ncbi.nlm.nih.gov/pubmed/37289023 http://dx.doi.org/10.1002/pro.4695 |
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author | Kovvali, Sravya Gao, Yuan Cool, Austin Lindert, Steffen Wysocki, Vicki H. Bell, Charles E. Gopalan, Venkat |
author_facet | Kovvali, Sravya Gao, Yuan Cool, Austin Lindert, Steffen Wysocki, Vicki H. Bell, Charles E. Gopalan, Venkat |
author_sort | Kovvali, Sravya |
collection | PubMed |
description | Amadori rearrangement products are stable sugar‐amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose‐lysine (F‐Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F‐Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6‐phosphofructose‐lysine (6‐P‐F‐Lys). FrlB, a deglycase, then converts 6‐P‐F‐Lys to L‐lysine and glucose‐6‐phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8‐Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6‐P‐F‐Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine‐6‐phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB—6‐P‐F‐Lys on GlmS—fructose‐6‐phosphate structures revealed parallels in their active‐site arrangement and guided our selection of seven putative active‐site residues in FrlB for site‐directed mutagenesis. Activity assays with eight recombinant single‐substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface‐induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x‐ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure–function and mechanistic studies of enzymes. |
format | Online Article Text |
id | pubmed-10285752 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | John Wiley & Sons, Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-102857522023-07-01 Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine Kovvali, Sravya Gao, Yuan Cool, Austin Lindert, Steffen Wysocki, Vicki H. Bell, Charles E. Gopalan, Venkat Protein Sci Articles Amadori rearrangement products are stable sugar‐amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose‐lysine (F‐Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F‐Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6‐phosphofructose‐lysine (6‐P‐F‐Lys). FrlB, a deglycase, then converts 6‐P‐F‐Lys to L‐lysine and glucose‐6‐phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8‐Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6‐P‐F‐Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine‐6‐phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB—6‐P‐F‐Lys on GlmS—fructose‐6‐phosphate structures revealed parallels in their active‐site arrangement and guided our selection of seven putative active‐site residues in FrlB for site‐directed mutagenesis. Activity assays with eight recombinant single‐substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface‐induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x‐ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure–function and mechanistic studies of enzymes. John Wiley & Sons, Inc. 2023-07-01 /pmc/articles/PMC10285752/ /pubmed/37289023 http://dx.doi.org/10.1002/pro.4695 Text en © 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Articles Kovvali, Sravya Gao, Yuan Cool, Austin Lindert, Steffen Wysocki, Vicki H. Bell, Charles E. Gopalan, Venkat Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine |
title | Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine |
title_full | Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine |
title_fullStr | Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine |
title_full_unstemmed | Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine |
title_short | Insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine |
title_sort | insights into the catalytic mechanism of a bacterial deglycase essential for utilization of fructose‐lysine |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10285752/ https://www.ncbi.nlm.nih.gov/pubmed/37289023 http://dx.doi.org/10.1002/pro.4695 |
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