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Structure determination and functional analysis of a chromate reductase from Gluconacetobacter hansenii.

Jin H, Zhang Y, Buchko GW, Varnum SM, Robinson H, Squier TC, Long PE - PLoS ONE (2012)

Bottom Line: Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions.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.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States of America. hongjunj@mir.wustl.edu

ABSTRACT
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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Structure proximal to bound FMN.A. Electron density surrounding FMN and chloride ion (gray sphere) contoured at 1.0 σ. B. Schematic representation of hydrophobic contacts (arc with radiating spokes) and potential hydrogen bonds (dashed lines) between FMN and two monomeric units (chain A and C) of the Gh-ChrR tetramer. Atoms are color-coded: black = carbon, red = oxygen, blue = nitrogen. This image was produced using the program LIGPLOT[62].
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pone-0042432-g003: Structure proximal to bound FMN.A. Electron density surrounding FMN and chloride ion (gray sphere) contoured at 1.0 σ. B. Schematic representation of hydrophobic contacts (arc with radiating spokes) and potential hydrogen bonds (dashed lines) between FMN and two monomeric units (chain A and C) of the Gh-ChrR tetramer. Atoms are color-coded: black = carbon, red = oxygen, blue = nitrogen. This image was produced using the program LIGPLOT[62].

Mentions: As shown in Figure 2A and 2B, Gh-ChrR crystallized with one molecule of FMN associated with each protein monomer. FMN binds in a pocket on the surface of Gh-ChrR near the dimer interface. The FMN binding pocket is more clearly illustrated in Figure 2C, which highlights the electrostatic potential at the solvent-accessible surface of Gh-ChrR. The negatively charged ribityl phosphate group of FMN is deeply buried in a positively charged region (blue) composed of residues in the loop between β1 and α1 (S15–N22) and a positive electrostatic dipole from the N-terminus of the capped α-helix (α1) similar to that previously reported [29]. On the other hand, the aromatic isoalloxazine ring sits in a more hydrophobic region (white) of the binding pocket. Details of the protein-FMN contacts responsible for stabilizing the complex are shown schematically in two-dimensions in Figure 3: 12 hydrogen bonds and five hydrophobic contacts. Except for two hydrophobic contacts (Y51’ and R101’), the FMN-protein interactions at the dimer interface are with one monomeric unit. As a consequence, the active site of Gh-ChrR is open and solvent accessible, a feature observed at the active site of oxidoreductases that facilitates promiscuous exchange of substrates [30]–[32].


Structure determination and functional analysis of a chromate reductase from Gluconacetobacter hansenii.

Jin H, Zhang Y, Buchko GW, Varnum SM, Robinson H, Squier TC, Long PE - PLoS ONE (2012)

Structure proximal to bound FMN.A. Electron density surrounding FMN and chloride ion (gray sphere) contoured at 1.0 σ. B. Schematic representation of hydrophobic contacts (arc with radiating spokes) and potential hydrogen bonds (dashed lines) between FMN and two monomeric units (chain A and C) of the Gh-ChrR tetramer. Atoms are color-coded: black = carbon, red = oxygen, blue = nitrogen. This image was produced using the program LIGPLOT[62].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412864&req=5

pone-0042432-g003: Structure proximal to bound FMN.A. Electron density surrounding FMN and chloride ion (gray sphere) contoured at 1.0 σ. B. Schematic representation of hydrophobic contacts (arc with radiating spokes) and potential hydrogen bonds (dashed lines) between FMN and two monomeric units (chain A and C) of the Gh-ChrR tetramer. Atoms are color-coded: black = carbon, red = oxygen, blue = nitrogen. This image was produced using the program LIGPLOT[62].
Mentions: As shown in Figure 2A and 2B, Gh-ChrR crystallized with one molecule of FMN associated with each protein monomer. FMN binds in a pocket on the surface of Gh-ChrR near the dimer interface. The FMN binding pocket is more clearly illustrated in Figure 2C, which highlights the electrostatic potential at the solvent-accessible surface of Gh-ChrR. The negatively charged ribityl phosphate group of FMN is deeply buried in a positively charged region (blue) composed of residues in the loop between β1 and α1 (S15–N22) and a positive electrostatic dipole from the N-terminus of the capped α-helix (α1) similar to that previously reported [29]. On the other hand, the aromatic isoalloxazine ring sits in a more hydrophobic region (white) of the binding pocket. Details of the protein-FMN contacts responsible for stabilizing the complex are shown schematically in two-dimensions in Figure 3: 12 hydrogen bonds and five hydrophobic contacts. Except for two hydrophobic contacts (Y51’ and R101’), the FMN-protein interactions at the dimer interface are with one monomeric unit. As a consequence, the active site of Gh-ChrR is open and solvent accessible, a feature observed at the active site of oxidoreductases that facilitates promiscuous exchange of substrates [30]–[32].

Bottom Line: Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions.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.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States of America. hongjunj@mir.wustl.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus