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Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii

Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal redu...

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Autores principales: Jin, Hongjun, Zhang, Yanfeng, Buchko, Garry W., Varnum, Susan M., Robinson, Howard, Squier, Thomas C., Long, Philip E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412864/
https://www.ncbi.nlm.nih.gov/pubmed/22879982
http://dx.doi.org/10.1371/journal.pone.0042432
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author Jin, Hongjun
Zhang, Yanfeng
Buchko, Garry W.
Varnum, Susan M.
Robinson, Howard
Squier, Thomas C.
Long, Philip E.
author_facet Jin, Hongjun
Zhang, Yanfeng
Buchko, Garry W.
Varnum, Susan M.
Robinson, Howard
Squier, Thomas C.
Long, Philip E.
author_sort Jin, Hongjun
collection PubMed
description Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from Gluconacetobacter hansenii (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90–95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation.
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spelling pubmed-34128642012-08-09 Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii Jin, Hongjun Zhang, Yanfeng Buchko, Garry W. Varnum, Susan M. Robinson, Howard Squier, Thomas C. Long, Philip E. PLoS One Research Article Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from Gluconacetobacter hansenii (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90–95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation. Public Library of Science 2012-08-06 /pmc/articles/PMC3412864/ /pubmed/22879982 http://dx.doi.org/10.1371/journal.pone.0042432 Text en © 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Jin, Hongjun
Zhang, Yanfeng
Buchko, Garry W.
Varnum, Susan M.
Robinson, Howard
Squier, Thomas C.
Long, Philip E.
Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii
title Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii
title_full Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii
title_fullStr Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii
title_full_unstemmed Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii
title_short Structure Determination and Functional Analysis of a Chromate Reductase from Gluconacetobacter hansenii
title_sort structure determination and functional analysis of a chromate reductase from gluconacetobacter hansenii
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412864/
https://www.ncbi.nlm.nih.gov/pubmed/22879982
http://dx.doi.org/10.1371/journal.pone.0042432
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