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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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Substrate inhibition by NADH in an ordered bireactant mechanism.A. Double-reciprocal plots of initial velocities versus substrate concentrations assayed with fixed concentration of NADH: 9.5 µM (open diamond), 19 µM (closed diamond), 25 µM (open triangle), 37.5 µM (closed triangle), 50 µM (open square), 75 µM (closed square), 100 µM (open circle), and 200 µM (closed circle). The VMax is calculated based on the y-axis intercept on this plot. B. Relationship between the slopes (i.e., Slope 1/CrO42−) in Figure 1A at each of seven fixed NADH concentrations. C. Double-reciprocal plots of initial velocities versus substrate concentrations with fixed concentration of CrO42−: 31 µM (open triangle), 62 µM (closed triangle), 125 µM (open square), 250 µM (closed square), 500 µM (open circle), and 1000 µM (closed circle). At low NADH concentrations it is possible to fit the data with a straight line. However, at high NADH concentrations, individual curves bend upwards. Values for KmA, KmB, Kia and Ki were calculated from axes-intercepts and slopes in panels B and C (see Table S2) [20]. D. Cleland notation depicting catalytic mechanism of Gh-ChrR, showing substrate inhibition by NADH binding to FMN-E to form a dead-end complex FMN-E-NADH that competes with metal complex formation, Mox-FMNH2-E-NADH.
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pone-0042432-g001: Substrate inhibition by NADH in an ordered bireactant mechanism.A. Double-reciprocal plots of initial velocities versus substrate concentrations assayed with fixed concentration of NADH: 9.5 µM (open diamond), 19 µM (closed diamond), 25 µM (open triangle), 37.5 µM (closed triangle), 50 µM (open square), 75 µM (closed square), 100 µM (open circle), and 200 µM (closed circle). The VMax is calculated based on the y-axis intercept on this plot. B. Relationship between the slopes (i.e., Slope 1/CrO42−) in Figure 1A at each of seven fixed NADH concentrations. C. Double-reciprocal plots of initial velocities versus substrate concentrations with fixed concentration of CrO42−: 31 µM (open triangle), 62 µM (closed triangle), 125 µM (open square), 250 µM (closed square), 500 µM (open circle), and 1000 µM (closed circle). At low NADH concentrations it is possible to fit the data with a straight line. However, at high NADH concentrations, individual curves bend upwards. Values for KmA, KmB, Kia and Ki were calculated from axes-intercepts and slopes in panels B and C (see Table S2) [20]. D. Cleland notation depicting catalytic mechanism of Gh-ChrR, showing substrate inhibition by NADH binding to FMN-E to form a dead-end complex FMN-E-NADH that competes with metal complex formation, Mox-FMNH2-E-NADH.

Mentions: Initial-velocity measurements with chromate as the substrate and NADH as the electron donor were carried out at a fixed enzyme concentration. Consistent with a mechanism involving substrate inhibition by NADH, there were substantial reductions in initial enzyme velocities upon increasing NADH concentrations at fixed chromate concentrations (Figure 1A). Other mechanisms, such as those involving a bi-bi ping pong reaction mechanism where increasing concentrations of NADH results in enhancements in enzyme velocity, are not consistent with the experimental data [20].


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)

Substrate inhibition by NADH in an ordered bireactant mechanism.A. Double-reciprocal plots of initial velocities versus substrate concentrations assayed with fixed concentration of NADH: 9.5 µM (open diamond), 19 µM (closed diamond), 25 µM (open triangle), 37.5 µM (closed triangle), 50 µM (open square), 75 µM (closed square), 100 µM (open circle), and 200 µM (closed circle). The VMax is calculated based on the y-axis intercept on this plot. B. Relationship between the slopes (i.e., Slope 1/CrO42−) in Figure 1A at each of seven fixed NADH concentrations. C. Double-reciprocal plots of initial velocities versus substrate concentrations with fixed concentration of CrO42−: 31 µM (open triangle), 62 µM (closed triangle), 125 µM (open square), 250 µM (closed square), 500 µM (open circle), and 1000 µM (closed circle). At low NADH concentrations it is possible to fit the data with a straight line. However, at high NADH concentrations, individual curves bend upwards. Values for KmA, KmB, Kia and Ki were calculated from axes-intercepts and slopes in panels B and C (see Table S2) [20]. D. Cleland notation depicting catalytic mechanism of Gh-ChrR, showing substrate inhibition by NADH binding to FMN-E to form a dead-end complex FMN-E-NADH that competes with metal complex formation, Mox-FMNH2-E-NADH.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042432-g001: Substrate inhibition by NADH in an ordered bireactant mechanism.A. Double-reciprocal plots of initial velocities versus substrate concentrations assayed with fixed concentration of NADH: 9.5 µM (open diamond), 19 µM (closed diamond), 25 µM (open triangle), 37.5 µM (closed triangle), 50 µM (open square), 75 µM (closed square), 100 µM (open circle), and 200 µM (closed circle). The VMax is calculated based on the y-axis intercept on this plot. B. Relationship between the slopes (i.e., Slope 1/CrO42−) in Figure 1A at each of seven fixed NADH concentrations. C. Double-reciprocal plots of initial velocities versus substrate concentrations with fixed concentration of CrO42−: 31 µM (open triangle), 62 µM (closed triangle), 125 µM (open square), 250 µM (closed square), 500 µM (open circle), and 1000 µM (closed circle). At low NADH concentrations it is possible to fit the data with a straight line. However, at high NADH concentrations, individual curves bend upwards. Values for KmA, KmB, Kia and Ki were calculated from axes-intercepts and slopes in panels B and C (see Table S2) [20]. D. Cleland notation depicting catalytic mechanism of Gh-ChrR, showing substrate inhibition by NADH binding to FMN-E to form a dead-end complex FMN-E-NADH that competes with metal complex formation, Mox-FMNH2-E-NADH.
Mentions: Initial-velocity measurements with chromate as the substrate and NADH as the electron donor were carried out at a fixed enzyme concentration. Consistent with a mechanism involving substrate inhibition by NADH, there were substantial reductions in initial enzyme velocities upon increasing NADH concentrations at fixed chromate concentrations (Figure 1A). Other mechanisms, such as those involving a bi-bi ping pong reaction mechanism where increasing concentrations of NADH results in enhancements in enzyme velocity, are not consistent with the experimental data [20].

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