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Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters

[Image: see text] The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to...

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Autores principales: Xiang, Dao Feng, Kumaran, Desigan, Swaminathan, Subramanyam, Raushel, Frank M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045322/
https://www.ncbi.nlm.nih.gov/pubmed/24832101
http://dx.doi.org/10.1021/bi5004266
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author Xiang, Dao Feng
Kumaran, Desigan
Swaminathan, Subramanyam
Raushel, Frank M.
author_facet Xiang, Dao Feng
Kumaran, Desigan
Swaminathan, Subramanyam
Raushel, Frank M.
author_sort Xiang, Dao Feng
collection PubMed
description [Image: see text] The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)(8)-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (k(cat)/K(m) = 1.0 × 10(5) M(–1) s(–1)), 2-naphthyl propionate (k(cat)/K(m) = 1.5 × 10(5) M(–1) s(–1)), 1-naphthyl acetate (k(cat)/K(m) = 7.5 × 10(3) M(–1) s(–1)), 4-methylumbelliferyl acetate (k(cat)/K(m) = 2.7 × 10(3) M(–1) s(–1)), 4-nitrophenyl acetate (k(cat)/K(m) = 2.3 × 10(5) M(–1) s(–1)), and 4-nitrophenyl butyrate (k(cat)/K(m) = 8.8 × 10(5) M(–1) s(–1)). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (k(cat)/K(m) = 3.8 × 10(5) M(–1) s(–1)) and isobutyl 4-nitrophenyl methylphosphonate (k(cat)/K(m) = 1.1 × 10(4) M(–1) s(–1)). The (S(P))-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (R(P))-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations.
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spelling pubmed-40453222015-05-15 Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters Xiang, Dao Feng Kumaran, Desigan Swaminathan, Subramanyam Raushel, Frank M. Biochemistry [Image: see text] The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)(8)-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (k(cat)/K(m) = 1.0 × 10(5) M(–1) s(–1)), 2-naphthyl propionate (k(cat)/K(m) = 1.5 × 10(5) M(–1) s(–1)), 1-naphthyl acetate (k(cat)/K(m) = 7.5 × 10(3) M(–1) s(–1)), 4-methylumbelliferyl acetate (k(cat)/K(m) = 2.7 × 10(3) M(–1) s(–1)), 4-nitrophenyl acetate (k(cat)/K(m) = 2.3 × 10(5) M(–1) s(–1)), and 4-nitrophenyl butyrate (k(cat)/K(m) = 8.8 × 10(5) M(–1) s(–1)). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (k(cat)/K(m) = 3.8 × 10(5) M(–1) s(–1)) and isobutyl 4-nitrophenyl methylphosphonate (k(cat)/K(m) = 1.1 × 10(4) M(–1) s(–1)). The (S(P))-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (R(P))-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations. American Chemical Society 2014-05-15 2014-06-03 /pmc/articles/PMC4045322/ /pubmed/24832101 http://dx.doi.org/10.1021/bi5004266 Text en Copyright © 2014 American Chemical Society
spellingShingle Xiang, Dao Feng
Kumaran, Desigan
Swaminathan, Subramanyam
Raushel, Frank M.
Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters
title Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters
title_full Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters
title_fullStr Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters
title_full_unstemmed Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters
title_short Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters
title_sort structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045322/
https://www.ncbi.nlm.nih.gov/pubmed/24832101
http://dx.doi.org/10.1021/bi5004266
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