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Nickel quercetinase, a "promiscuous" metalloenzyme: metal incorporation and metal ligand substitution studies.

Nianios D, Thierbach S, Steimer L, Lulchev P, Klostermeier D, Fetzner S - BMC Biochem. (2015)

Bottom Line: Iron, in contrast to the other metals, does not support catalytic activity.Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity.The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstrasse 3, Muenster, D-48149, Germany. d_nian01@uni-muenster.de.

ABSTRACT

Background: Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni(2+) and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant.

Results: Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni(2+) was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni(2+) ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity.

Conclusions: Heterologous formation of catalytically active, native QueD holoenzyme requires Ni(2+), Co(2+) or Mn(2+), i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni(2+) most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.

No MeSH data available.


Related in: MedlinePlus

Far-UV CD spectra of QueD variants carrying amino acid replacements. (A), Ni-QueD proteins with amino acid substitutions in the 3His/1Glu motif proposed to ligate the metal ion, and (B) Co-QueD with a substitution of the glutamate ligand by histidine. Protein samples in 10 mM potassium phosphate buffer (pH 8.0) were adjusted to ~10 μM (based on measuring absorbance at 280 nm). Spectra were recorded at 25°C in a 1 mm path length cell; bandwidth: 0.1 nm.
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Fig3: Far-UV CD spectra of QueD variants carrying amino acid replacements. (A), Ni-QueD proteins with amino acid substitutions in the 3His/1Glu motif proposed to ligate the metal ion, and (B) Co-QueD with a substitution of the glutamate ligand by histidine. Protein samples in 10 mM potassium phosphate buffer (pH 8.0) were adjusted to ~10 μM (based on measuring absorbance at 280 nm). Spectra were recorded at 25°C in a 1 mm path length cell; bandwidth: 0.1 nm.

Mentions: To analyze the role of the metal-ligating amino acids for the structural integrity and function of Ni-QueD, the individual residues of the 3His/1Glu motif (Figure 1) were replaced by site-directed mutagenesis, and the QueD proteins produced by the respective E. coli strain grown in Ni2+-supplemented medium were purified and characterized with respect to metal contents, catalytic activity, and ability to bind quercetin (Table 2). All variants, with the exception of the H69A substitution, showed a decrease in nickel contents compared to the wild-type protein, and a relative increase in the zinc contents pointing towards a somewhat relaxed metal specificity. The KD values of the protein-quercetin complexes show that all protein variants retained a high affinity for the organic substrate. Replacement of the histidine residues at position 69 or 115 with alanine resulted in catalytically inactive protein variants, whereas the QueD-H71A and -E76D proteins retained marginal activity (Table 2). The CD spectra of the protein variants showed an increased intensity of the negative band (at approx. 215 nm) and a hypsochromic (blue) shift of the positive band (<200 nm) compared to the wild type protein (Figure 3A). Thus, the loss of activity could be due to perturbation of the secondary structure.Table 2


Nickel quercetinase, a "promiscuous" metalloenzyme: metal incorporation and metal ligand substitution studies.

Nianios D, Thierbach S, Steimer L, Lulchev P, Klostermeier D, Fetzner S - BMC Biochem. (2015)

Far-UV CD spectra of QueD variants carrying amino acid replacements. (A), Ni-QueD proteins with amino acid substitutions in the 3His/1Glu motif proposed to ligate the metal ion, and (B) Co-QueD with a substitution of the glutamate ligand by histidine. Protein samples in 10 mM potassium phosphate buffer (pH 8.0) were adjusted to ~10 μM (based on measuring absorbance at 280 nm). Spectra were recorded at 25°C in a 1 mm path length cell; bandwidth: 0.1 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4416304&req=5

Fig3: Far-UV CD spectra of QueD variants carrying amino acid replacements. (A), Ni-QueD proteins with amino acid substitutions in the 3His/1Glu motif proposed to ligate the metal ion, and (B) Co-QueD with a substitution of the glutamate ligand by histidine. Protein samples in 10 mM potassium phosphate buffer (pH 8.0) were adjusted to ~10 μM (based on measuring absorbance at 280 nm). Spectra were recorded at 25°C in a 1 mm path length cell; bandwidth: 0.1 nm.
Mentions: To analyze the role of the metal-ligating amino acids for the structural integrity and function of Ni-QueD, the individual residues of the 3His/1Glu motif (Figure 1) were replaced by site-directed mutagenesis, and the QueD proteins produced by the respective E. coli strain grown in Ni2+-supplemented medium were purified and characterized with respect to metal contents, catalytic activity, and ability to bind quercetin (Table 2). All variants, with the exception of the H69A substitution, showed a decrease in nickel contents compared to the wild-type protein, and a relative increase in the zinc contents pointing towards a somewhat relaxed metal specificity. The KD values of the protein-quercetin complexes show that all protein variants retained a high affinity for the organic substrate. Replacement of the histidine residues at position 69 or 115 with alanine resulted in catalytically inactive protein variants, whereas the QueD-H71A and -E76D proteins retained marginal activity (Table 2). The CD spectra of the protein variants showed an increased intensity of the negative band (at approx. 215 nm) and a hypsochromic (blue) shift of the positive band (<200 nm) compared to the wild type protein (Figure 3A). Thus, the loss of activity could be due to perturbation of the secondary structure.Table 2

Bottom Line: Iron, in contrast to the other metals, does not support catalytic activity.Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity.The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstrasse 3, Muenster, D-48149, Germany. d_nian01@uni-muenster.de.

ABSTRACT

Background: Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni(2+) and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant.

Results: Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni(2+) was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni(2+) ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity.

Conclusions: Heterologous formation of catalytically active, native QueD holoenzyme requires Ni(2+), Co(2+) or Mn(2+), i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni(2+) most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.

No MeSH data available.


Related in: MedlinePlus