Cargando…
Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus
The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradatio...
Autores principales: | , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099567/ https://www.ncbi.nlm.nih.gov/pubmed/32266226 http://dx.doi.org/10.3389/fbioe.2020.00185 |
_version_ | 1783511331982278656 |
---|---|
author | Krevet, Sabine Shen, Lu Bohnen, Timon Schoenenberger, Bernhard Meier, Roland Obkircher, Markus Bangert, Klara Koehling, Rudolf Allenspach, Eric Wohlgemuth, Roland Siebers, Bettina Bräsen, Christopher |
author_facet | Krevet, Sabine Shen, Lu Bohnen, Timon Schoenenberger, Bernhard Meier, Roland Obkircher, Markus Bangert, Klara Koehling, Rudolf Allenspach, Eric Wohlgemuth, Roland Siebers, Bettina Bräsen, Christopher |
author_sort | Krevet, Sabine |
collection | PubMed |
description | The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a V(max) of 61.6 U mg(–1) and a K(M) of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses. |
format | Online Article Text |
id | pubmed-7099567 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-70995672020-04-07 Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus Krevet, Sabine Shen, Lu Bohnen, Timon Schoenenberger, Bernhard Meier, Roland Obkircher, Markus Bangert, Klara Koehling, Rudolf Allenspach, Eric Wohlgemuth, Roland Siebers, Bettina Bräsen, Christopher Front Bioeng Biotechnol Bioengineering and Biotechnology The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a V(max) of 61.6 U mg(–1) and a K(M) of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses. Frontiers Media S.A. 2020-03-20 /pmc/articles/PMC7099567/ /pubmed/32266226 http://dx.doi.org/10.3389/fbioe.2020.00185 Text en Copyright © 2020 Krevet, Shen, Bohnen, Schoenenberger, Meier, Obkircher, Bangert, Koehling, Allenspach, Wohlgemuth, Siebers and Bräsen. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Krevet, Sabine Shen, Lu Bohnen, Timon Schoenenberger, Bernhard Meier, Roland Obkircher, Markus Bangert, Klara Koehling, Rudolf Allenspach, Eric Wohlgemuth, Roland Siebers, Bettina Bräsen, Christopher Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus |
title | Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus |
title_full | Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus |
title_fullStr | Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus |
title_full_unstemmed | Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus |
title_short | Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus |
title_sort | enzymatic synthesis of 2-keto-3-deoxy-6-phosphogluconate by the 6-phosphogluconate-dehydratase from caulobacter crescentus |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099567/ https://www.ncbi.nlm.nih.gov/pubmed/32266226 http://dx.doi.org/10.3389/fbioe.2020.00185 |
work_keys_str_mv | AT krevetsabine enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT shenlu enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT bohnentimon enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT schoenenbergerbernhard enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT meierroland enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT obkirchermarkus enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT bangertklara enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT koehlingrudolf enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT allenspacheric enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT wohlgemuthroland enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT siebersbettina enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus AT brasenchristopher enzymaticsynthesisof2keto3deoxy6phosphogluconatebythe6phosphogluconatedehydratasefromcaulobactercrescentus |