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Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling
In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆(1)-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10537722/ https://www.ncbi.nlm.nih.gov/pubmed/37764979 http://dx.doi.org/10.3390/pathogens12091171 |
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author | Kumar, Santosh Sega, Steven Lynn-Barbe, Jamie K. Harris, Dannika L. Koehn, Jordan T. Crans, Debbie C. Crick, Dean C. |
author_facet | Kumar, Santosh Sega, Steven Lynn-Barbe, Jamie K. Harris, Dannika L. Koehn, Jordan T. Crans, Debbie C. Crick, Dean C. |
author_sort | Kumar, Santosh |
collection | PubMed |
description | In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆(1)-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H(2))] specificity constants K(cat)/K(m) were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A K(m) value of 120 ± 0.015 µM was determined for P5C, while the K(m) value for NAD(+) was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity. |
format | Online Article Text |
id | pubmed-10537722 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-105377222023-09-29 Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling Kumar, Santosh Sega, Steven Lynn-Barbe, Jamie K. Harris, Dannika L. Koehn, Jordan T. Crans, Debbie C. Crick, Dean C. Pathogens Article In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆(1)-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H(2))] specificity constants K(cat)/K(m) were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A K(m) value of 120 ± 0.015 µM was determined for P5C, while the K(m) value for NAD(+) was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity. MDPI 2023-09-18 /pmc/articles/PMC10537722/ /pubmed/37764979 http://dx.doi.org/10.3390/pathogens12091171 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Kumar, Santosh Sega, Steven Lynn-Barbe, Jamie K. Harris, Dannika L. Koehn, Jordan T. Crans, Debbie C. Crick, Dean C. Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling |
title | Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling |
title_full | Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling |
title_fullStr | Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling |
title_full_unstemmed | Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling |
title_short | Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling |
title_sort | proline dehydrogenase and pyrroline 5 carboxylate dehydrogenase from mycobacterium tuberculosis: evidence for substrate channeling |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10537722/ https://www.ncbi.nlm.nih.gov/pubmed/37764979 http://dx.doi.org/10.3390/pathogens12091171 |
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