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...

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Main Authors:	Raj, Hans, Puthan Veetil, Vinod, Szymanski, Wiktor, Dekker, Frank J., Quax, Wim J., Feringa, Ben L., Janssen, Dick B., Poelarends, Gerrit J.
Format:	Online
Language:	English
Published:	Springer-Verlag 2011
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310078/

id	pubmed-3310078
recordtype	oai_dc
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. 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 Mg2+ 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 (kcat = 78 s−1 and Km = 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. Springer-Verlag 2011-10-18 2012-04 /pmc/articles/PMC3310078/ /pubmed/22005738 http://dx.doi.org/10.1007/s00253-011-3615-6 Text en © The Author(s) 2011
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Raj, Hans Puthan Veetil, Vinod Szymanski, Wiktor Dekker, Frank J. Quax, Wim J. Feringa, Ben L. Janssen, Dick B. Poelarends, Gerrit J.
spellingShingle	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
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
title	Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans
title_short	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_sort	characterization of a thermostable methylaspartate ammonia lyase from carboxydothermus hydrogenoformans
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 Mg2+ 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 (kcat = 78 s−1 and Km = 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.
publisher	Springer-Verlag
publishDate	2011
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310078/
_version_	1611516061715267584

Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans

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