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Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans
Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substitute...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer-Verlag
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310078/ https://www.ncbi.nlm.nih.gov/pubmed/22005738 http://dx.doi.org/10.1007/s00253-011-3615-6 |
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author | Raj, Hans Puthan Veetil, Vinod Szymanski, Wiktor Dekker, Frank J. Quax, Wim J. Feringa, Ben L. Janssen, Dick B. Poelarends, Gerrit J. |
author_facet | Raj, Hans Puthan Veetil, Vinod Szymanski, Wiktor Dekker, Frank J. Quax, Wim J. Feringa, Ben L. Janssen, Dick B. Poelarends, Gerrit J. |
author_sort | Raj, Hans |
collection | PubMed |
description | Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (∼49 kDa each), and requires Mg(2+) and K(+) ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k (cat) = 78 s(−1) and K (m) = 16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-011-3615-6) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-3310078 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Springer-Verlag |
record_format | MEDLINE/PubMed |
spelling | pubmed-33100782012-03-22 Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans Raj, Hans Puthan Veetil, Vinod Szymanski, Wiktor Dekker, Frank J. Quax, Wim J. Feringa, Ben L. Janssen, Dick B. Poelarends, Gerrit J. Appl Microbiol Biotechnol Biotechnologically Relevant Enzymes and Proteins Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (∼49 kDa each), and requires Mg(2+) and K(+) ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k (cat) = 78 s(−1) and K (m) = 16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-011-3615-6) contains supplementary material, which is available to authorized users. Springer-Verlag 2011-10-18 2012 /pmc/articles/PMC3310078/ /pubmed/22005738 http://dx.doi.org/10.1007/s00253-011-3615-6 Text en © The Author(s) 2011 https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. |
spellingShingle | Biotechnologically Relevant Enzymes and Proteins Raj, Hans Puthan Veetil, Vinod Szymanski, Wiktor Dekker, Frank J. Quax, Wim J. Feringa, Ben L. Janssen, Dick B. Poelarends, Gerrit J. Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans |
title | Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans |
title_full | Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans |
title_fullStr | Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans |
title_full_unstemmed | Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans |
title_short | Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans |
title_sort | characterization of a thermostable methylaspartate ammonia lyase from carboxydothermus hydrogenoformans |
topic | Biotechnologically Relevant Enzymes and Proteins |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310078/ https://www.ncbi.nlm.nih.gov/pubmed/22005738 http://dx.doi.org/10.1007/s00253-011-3615-6 |
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