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An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis
The biosynthesis of many vitamins and coenzymes has often proved difficult to elucidate due to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in E. coli, and by His-tagging the terminal enzyme...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480714/ https://www.ncbi.nlm.nih.gov/pubmed/23042036 http://dx.doi.org/10.1038/nchembio.1086 |
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author | Deery, Evelyne Schroeder, Susanne Lawrence, Andrew D. Taylor, Samantha L. Seyedarabi, Arefeh Waterman, Jitka Wilson, Keith S. Brown, David Geeves, Michael A. Howard, Mark J. Pickersgill, Richard W. Warren, Martin J. |
author_facet | Deery, Evelyne Schroeder, Susanne Lawrence, Andrew D. Taylor, Samantha L. Seyedarabi, Arefeh Waterman, Jitka Wilson, Keith S. Brown, David Geeves, Michael A. Howard, Mark J. Pickersgill, Richard W. Warren, Martin J. |
author_sort | Deery, Evelyne |
collection | PubMed |
description | The biosynthesis of many vitamins and coenzymes has often proved difficult to elucidate due to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in E. coli, and by His-tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly-bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalysed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, has been identified, which can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling. |
format | Online Article Text |
id | pubmed-3480714 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
record_format | MEDLINE/PubMed |
spelling | pubmed-34807142013-05-01 An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis Deery, Evelyne Schroeder, Susanne Lawrence, Andrew D. Taylor, Samantha L. Seyedarabi, Arefeh Waterman, Jitka Wilson, Keith S. Brown, David Geeves, Michael A. Howard, Mark J. Pickersgill, Richard W. Warren, Martin J. Nat Chem Biol Article The biosynthesis of many vitamins and coenzymes has often proved difficult to elucidate due to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B(12)) pathway in E. coli, and by His-tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly-bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalysed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, has been identified, which can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling. 2012-10-07 2012-11 /pmc/articles/PMC3480714/ /pubmed/23042036 http://dx.doi.org/10.1038/nchembio.1086 Text en Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms |
spellingShingle | Article Deery, Evelyne Schroeder, Susanne Lawrence, Andrew D. Taylor, Samantha L. Seyedarabi, Arefeh Waterman, Jitka Wilson, Keith S. Brown, David Geeves, Michael A. Howard, Mark J. Pickersgill, Richard W. Warren, Martin J. An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis |
title | An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis |
title_full | An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis |
title_fullStr | An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis |
title_full_unstemmed | An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis |
title_short | An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis |
title_sort | enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480714/ https://www.ncbi.nlm.nih.gov/pubmed/23042036 http://dx.doi.org/10.1038/nchembio.1086 |
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