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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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Putative Gh-ChrR NADH and substrate binding sites.A. NADH was modeled into the Gh-ChrR structure by superimposing it with the NADH-containing structure of EmoB (PDB entry: 2VZJ, Figure S7). The nicotinamide ring of NADH (primarily green stick model) is stacked on top of the isoalloxazine ring of FMN (primarily yellow stick model), and the adenosine part of NADH points to ribtyl group of FMN. The black arrow indicates the distance from C4N of NADH to the si-face of the FMN isoalloxazine ring. Residues N53, D54, E57, S100, R101 and F137 from chain A (cyan) and residues N85, P119, and T154 from chain C (gold) interact with NADH. B. The putative active site of Gh-ChrR shown with bound FMN (primarily yellow stick model) and a chloride ion (green sphere). The black arrow indicates the distance from the Cl− to the si-face of the FMN isoalloxazine ring. Key residue R101 holding chloride ion in place is shown in a stick model. Critical residues for hydride transfer, N85 and Y86 from chain A (cyan) and S118 from chain C (gold) are shown in a stick model. The green dash lines indicate the distance (∼3 Å) between N of amide group of N85/Y86 and O4, and the distance (∼3 Å) between OG of hydroxyl group of S118 and O2.
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pone-0042432-g004: Putative Gh-ChrR NADH and substrate binding sites.A. NADH was modeled into the Gh-ChrR structure by superimposing it with the NADH-containing structure of EmoB (PDB entry: 2VZJ, Figure S7). The nicotinamide ring of NADH (primarily green stick model) is stacked on top of the isoalloxazine ring of FMN (primarily yellow stick model), and the adenosine part of NADH points to ribtyl group of FMN. The black arrow indicates the distance from C4N of NADH to the si-face of the FMN isoalloxazine ring. Residues N53, D54, E57, S100, R101 and F137 from chain A (cyan) and residues N85, P119, and T154 from chain C (gold) interact with NADH. B. The putative active site of Gh-ChrR shown with bound FMN (primarily yellow stick model) and a chloride ion (green sphere). The black arrow indicates the distance from the Cl− to the si-face of the FMN isoalloxazine ring. Key residue R101 holding chloride ion in place is shown in a stick model. Critical residues for hydride transfer, N85 and Y86 from chain A (cyan) and S118 from chain C (gold) are shown in a stick model. The green dash lines indicate the distance (∼3 Å) between N of amide group of N85/Y86 and O4, and the distance (∼3 Å) between OG of hydroxyl group of S118 and O2.

Mentions: While bound FMN is observed in the crystal structure of Gh-ChrR (Figures 2 and 3), NADH, an essential electron transfer component of the reductive reactions catalyzed by NAD(P)H-dependent FMN reductases, is absent. Efforts to co-crystallize Gh-ChrR with NADH were unsuccessful. However, it is possible to predict the location of the NADH binding site on the FMN-Gh-ChrR structure by superposing it on the structure of a homologous NAD(P)H-dependent FMN reductase, EmoB from Mesorhizobium BNC1 complexed with FMN and NADH [28]. Both Gh-ChrR and EmoB form homotetramers that have similar structures for the individual monomeric subunits (RMSD =  2.6 Å with 161 aligned Cα atoms, Figure S8). In EmoB, the nicotinamide ring of NADH sits above the bound FMN and stacks against the isoalloxazine ring of FMN. Only two residues in EmoB were observed to interact with NADH, K81 and G112 [28]. In the superimposition with Gh-ChrR (Figure 4A and Figure S8), only one of the two equivalent residues, N85, is in a position to contact NADH, as G109 is too distant. The importance of N85 was confirmed by an N85A site-directed substitution (Table 2), resulting in an apparent Km value 3-fold larger than that for wild type Gh-ChrR that is consistent with a reduction in binding affinity. The aromatic ring of F137 is 2.82 Å from C4N of the NADH nicotinamide ring suggesting a possible hydrophobic interaction. Other Gh-ChrR residues that could potentially interact with NADH are N53, D54, and E57 at the adenosine part of NADH and P119 and T154 at the di-phosphate part of NADH. Collectively, the superposition of structures suggests that residues N53, D54, E57, S100, R101 and F137 from one monomer and residues N85, P119, and T154 from the other monomer of the dimer, may interact with NADH (Figure 4A), and further suggests that the active site of Gh-ChrR has ample room for NADH to enter.


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)

Putative Gh-ChrR NADH and substrate binding sites.A. NADH was modeled into the Gh-ChrR structure by superimposing it with the NADH-containing structure of EmoB (PDB entry: 2VZJ, Figure S7). The nicotinamide ring of NADH (primarily green stick model) is stacked on top of the isoalloxazine ring of FMN (primarily yellow stick model), and the adenosine part of NADH points to ribtyl group of FMN. The black arrow indicates the distance from C4N of NADH to the si-face of the FMN isoalloxazine ring. Residues N53, D54, E57, S100, R101 and F137 from chain A (cyan) and residues N85, P119, and T154 from chain C (gold) interact with NADH. B. The putative active site of Gh-ChrR shown with bound FMN (primarily yellow stick model) and a chloride ion (green sphere). The black arrow indicates the distance from the Cl− to the si-face of the FMN isoalloxazine ring. Key residue R101 holding chloride ion in place is shown in a stick model. Critical residues for hydride transfer, N85 and Y86 from chain A (cyan) and S118 from chain C (gold) are shown in a stick model. The green dash lines indicate the distance (∼3 Å) between N of amide group of N85/Y86 and O4, and the distance (∼3 Å) between OG of hydroxyl group of S118 and O2.
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Related In: Results  -  Collection

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

pone-0042432-g004: Putative Gh-ChrR NADH and substrate binding sites.A. NADH was modeled into the Gh-ChrR structure by superimposing it with the NADH-containing structure of EmoB (PDB entry: 2VZJ, Figure S7). The nicotinamide ring of NADH (primarily green stick model) is stacked on top of the isoalloxazine ring of FMN (primarily yellow stick model), and the adenosine part of NADH points to ribtyl group of FMN. The black arrow indicates the distance from C4N of NADH to the si-face of the FMN isoalloxazine ring. Residues N53, D54, E57, S100, R101 and F137 from chain A (cyan) and residues N85, P119, and T154 from chain C (gold) interact with NADH. B. The putative active site of Gh-ChrR shown with bound FMN (primarily yellow stick model) and a chloride ion (green sphere). The black arrow indicates the distance from the Cl− to the si-face of the FMN isoalloxazine ring. Key residue R101 holding chloride ion in place is shown in a stick model. Critical residues for hydride transfer, N85 and Y86 from chain A (cyan) and S118 from chain C (gold) are shown in a stick model. The green dash lines indicate the distance (∼3 Å) between N of amide group of N85/Y86 and O4, and the distance (∼3 Å) between OG of hydroxyl group of S118 and O2.
Mentions: While bound FMN is observed in the crystal structure of Gh-ChrR (Figures 2 and 3), NADH, an essential electron transfer component of the reductive reactions catalyzed by NAD(P)H-dependent FMN reductases, is absent. Efforts to co-crystallize Gh-ChrR with NADH were unsuccessful. However, it is possible to predict the location of the NADH binding site on the FMN-Gh-ChrR structure by superposing it on the structure of a homologous NAD(P)H-dependent FMN reductase, EmoB from Mesorhizobium BNC1 complexed with FMN and NADH [28]. Both Gh-ChrR and EmoB form homotetramers that have similar structures for the individual monomeric subunits (RMSD =  2.6 Å with 161 aligned Cα atoms, Figure S8). In EmoB, the nicotinamide ring of NADH sits above the bound FMN and stacks against the isoalloxazine ring of FMN. Only two residues in EmoB were observed to interact with NADH, K81 and G112 [28]. In the superimposition with Gh-ChrR (Figure 4A and Figure S8), only one of the two equivalent residues, N85, is in a position to contact NADH, as G109 is too distant. The importance of N85 was confirmed by an N85A site-directed substitution (Table 2), resulting in an apparent Km value 3-fold larger than that for wild type Gh-ChrR that is consistent with a reduction in binding affinity. The aromatic ring of F137 is 2.82 Å from C4N of the NADH nicotinamide ring suggesting a possible hydrophobic interaction. Other Gh-ChrR residues that could potentially interact with NADH are N53, D54, and E57 at the adenosine part of NADH and P119 and T154 at the di-phosphate part of NADH. Collectively, the superposition of structures suggests that residues N53, D54, E57, S100, R101 and F137 from one monomer and residues N85, P119, and T154 from the other monomer of the dimer, may interact with NADH (Figure 4A), and further suggests that the active site of Gh-ChrR has ample room for NADH to enter.

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