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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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Crystal structure of Gh-ChrR.Monomeric (A) and tetrameric (B) depictions of the 2.25 Å structure of Gh-ChrR showing the backbone fold, a space-filling model of bound FMN (elements color: red  =  oxygen, blue  =  nitrogen, gray  =  carbon) and bound chloride anion (green sphere). Secondary structural elements including the 310 helices (η) are numbered sequentially from the N-terminus. C. Electrostatic potentials at the solvent-accessible surface of Gh-ChrR. A stick model of the FMN molecule and the associated chloride ion (gray sphere) is highlighted. The electrostatic potential are drawn (Pymol) at a level of −71.817 kT/e (red) to +71.817 kT/e (blue), where k is the Boltzman’s constant, T is the absolute temperature, and e is the magnitude of the electron charge.
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pone-0042432-g002: Crystal structure of Gh-ChrR.Monomeric (A) and tetrameric (B) depictions of the 2.25 Å structure of Gh-ChrR showing the backbone fold, a space-filling model of bound FMN (elements color: red  =  oxygen, blue  =  nitrogen, gray  =  carbon) and bound chloride anion (green sphere). Secondary structural elements including the 310 helices (η) are numbered sequentially from the N-terminus. C. Electrostatic potentials at the solvent-accessible surface of Gh-ChrR. A stick model of the FMN molecule and the associated chloride ion (gray sphere) is highlighted. The electrostatic potential are drawn (Pymol) at a level of −71.817 kT/e (red) to +71.817 kT/e (blue), where k is the Boltzman’s constant, T is the absolute temperature, and e is the magnitude of the electron charge.

Mentions: The crystal structure of Gh-ChrR was elucidated to a resolution of 2.25 Å (Table 1). The crystallographic asymmetric unit contains four monomers, each with a single bound FMN. The tetrameric structure of Gh-ChrR is consistent with the result of size exclusion chromatography (∼80 kDa), as the mass of the monomeric unit is 21.3 kDa (193 native residues plus a six residue C-terminal poly-histidine tag) (Figure S7). A tetrameric oligomerization state was also recently reported for E. coli ChrR [23], a protein with 61% sequence identity to Gh-ChrR. For each monomer in the asymmetric unit, electron density is missing or uninterpretable for 5–6 residues at the N-terminus and 7–9 residues at the C-terminus. Aside from the residues near the termini, there are no significant conformational differences between the four monomers as the α-carbons of residues P6-T186 superimpose on each other with a RMSD ranging from 0.35 to 0.38 Å (UCSF-Chimeria) [24]. Figure 2A is a cartoon representation of the backbone fold for one of the four essentially identical monomers in the asymmetric unit with the elements of secondary structure labeled. Each monomer contains two 310-helices (I45-F48, V153-K156, labeled as η), six α-helices (F21-I32, Q53-E58, A62-T73, G90-R101, A125-L138, V167-T186) and five β-strands (L7-L13, I37-P40, A76-T81, P111-S118, A148-I150). The β-strands are organized into one parallel β-sheet, β2:β1:β3:β4:β5, flanked by helices α1 and α5 on one face and the remaining helices on the opposite face. The five longest helices are aligned in two groups that are approximately parallel to each other and orthogonal: (1) α1 and α3 and (2) α4, α5, and α6 so that helices are approximately parallel with each group and orthogonal between groups. Such a triple-layered, α/β/α structure resembles the fold in the flavodoxin superfamily of proteins [18], [25]. Such a triple-layered, α/β/α structure resembles the fold in the flavodoxin superfamily of proteins [19], [38] and is identical to the fold observed in the crystal structure recently reported for E.coli ChrR (PDB entry: 3SVL) [23], a structure that superimposes onto Gh-ChrR with a backbone RMSD of 0.9 Å.


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)

Crystal structure of Gh-ChrR.Monomeric (A) and tetrameric (B) depictions of the 2.25 Å structure of Gh-ChrR showing the backbone fold, a space-filling model of bound FMN (elements color: red  =  oxygen, blue  =  nitrogen, gray  =  carbon) and bound chloride anion (green sphere). Secondary structural elements including the 310 helices (η) are numbered sequentially from the N-terminus. C. Electrostatic potentials at the solvent-accessible surface of Gh-ChrR. A stick model of the FMN molecule and the associated chloride ion (gray sphere) is highlighted. The electrostatic potential are drawn (Pymol) at a level of −71.817 kT/e (red) to +71.817 kT/e (blue), where k is the Boltzman’s constant, T is the absolute temperature, and e is the magnitude of the electron charge.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042432-g002: Crystal structure of Gh-ChrR.Monomeric (A) and tetrameric (B) depictions of the 2.25 Å structure of Gh-ChrR showing the backbone fold, a space-filling model of bound FMN (elements color: red  =  oxygen, blue  =  nitrogen, gray  =  carbon) and bound chloride anion (green sphere). Secondary structural elements including the 310 helices (η) are numbered sequentially from the N-terminus. C. Electrostatic potentials at the solvent-accessible surface of Gh-ChrR. A stick model of the FMN molecule and the associated chloride ion (gray sphere) is highlighted. The electrostatic potential are drawn (Pymol) at a level of −71.817 kT/e (red) to +71.817 kT/e (blue), where k is the Boltzman’s constant, T is the absolute temperature, and e is the magnitude of the electron charge.
Mentions: The crystal structure of Gh-ChrR was elucidated to a resolution of 2.25 Å (Table 1). The crystallographic asymmetric unit contains four monomers, each with a single bound FMN. The tetrameric structure of Gh-ChrR is consistent with the result of size exclusion chromatography (∼80 kDa), as the mass of the monomeric unit is 21.3 kDa (193 native residues plus a six residue C-terminal poly-histidine tag) (Figure S7). A tetrameric oligomerization state was also recently reported for E. coli ChrR [23], a protein with 61% sequence identity to Gh-ChrR. For each monomer in the asymmetric unit, electron density is missing or uninterpretable for 5–6 residues at the N-terminus and 7–9 residues at the C-terminus. Aside from the residues near the termini, there are no significant conformational differences between the four monomers as the α-carbons of residues P6-T186 superimpose on each other with a RMSD ranging from 0.35 to 0.38 Å (UCSF-Chimeria) [24]. Figure 2A is a cartoon representation of the backbone fold for one of the four essentially identical monomers in the asymmetric unit with the elements of secondary structure labeled. Each monomer contains two 310-helices (I45-F48, V153-K156, labeled as η), six α-helices (F21-I32, Q53-E58, A62-T73, G90-R101, A125-L138, V167-T186) and five β-strands (L7-L13, I37-P40, A76-T81, P111-S118, A148-I150). The β-strands are organized into one parallel β-sheet, β2:β1:β3:β4:β5, flanked by helices α1 and α5 on one face and the remaining helices on the opposite face. The five longest helices are aligned in two groups that are approximately parallel to each other and orthogonal: (1) α1 and α3 and (2) α4, α5, and α6 so that helices are approximately parallel with each group and orthogonal between groups. Such a triple-layered, α/β/α structure resembles the fold in the flavodoxin superfamily of proteins [18], [25]. Such a triple-layered, α/β/α structure resembles the fold in the flavodoxin superfamily of proteins [19], [38] and is identical to the fold observed in the crystal structure recently reported for E.coli ChrR (PDB entry: 3SVL) [23], a structure that superimposes onto Gh-ChrR with a backbone RMSD of 0.9 Å.

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