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Crystal structure of the γ-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. strainWBC-3, a key enzyme involved in para-Nitrophenol degradation.

Su J, Zhang C, Zhang JJ, Wei T, Zhu D, Zhou NY, Gu Lc - BMC Struct. Biol. (2013)

Bottom Line: In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde.Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified.The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan 250100, China. n.zhou@pentium.whiov.ac.cn.

ABSTRACT

Background: para-Nitrophenol (PNP) is a highly toxic compound with threats to mammalian health. The pnpE-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in Pseudomonas sp. strain WBC-3, playing a key role in the catabolism of PNP to Krebs cycle intermediates. However, the catalyzing mechanism by PnpE has not been well understood.

Results: Here we report the crystal structures of the apo and NAD bound PnpE. In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other.

Conclusions: Six amino acids have interactions with the cofactor. They are C281, E247, Q210, W148, I146 and K172. Highly conserved residues C281 and E247 were identified to be critical for its catalytic activity. In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.

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Substrate binding pocket predicted with the autodock result. A. PnpE is shown in surface model in yellow. NAD is depicted in green sticks and γ-hydroxymuconic semialdehyde is shown in red sticks. B. PnpE is shown in green ribbon model. There are nine amino acids interact with the substrate: C281, E247, F150, I282, F154, H275, F447, W157 and N149.
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Figure 4: Substrate binding pocket predicted with the autodock result. A. PnpE is shown in surface model in yellow. NAD is depicted in green sticks and γ-hydroxymuconic semialdehyde is shown in red sticks. B. PnpE is shown in green ribbon model. There are nine amino acids interact with the substrate: C281, E247, F150, I282, F154, H275, F447, W157 and N149.

Mentions: The substrate γ-hydroxymuconic semialdehyde for PnpE contains an enol structure, which tends to convert into keto under aerobic condition. So it is impracticable to get holo-PnpE structure. To figure out the catalytic mechanism of PnpE without holo-PnpE structure we performed flexible docking for NAD bound PnpE and γ-hydroxymuconic semialdehyde using AUTODOCK (experimental procedure detailed in section 2.7). The docking result is illustrated in Figure 4. The substrate located in a narrow cleft close to NAD. There are nine residues (C281, E247, F150, I282, F154, H275, F447, W157, N149) probably involved in the substrate binding. C281 forms hydrogen bond with the aldehyde group of the substrate directly and may play a key role in the enzymatic reaction. F150, F154, F447, W157, N149, I282 and H275 probably composed a hydrophobic pocket to accommodate the substrate. However, W157 and F447 are a little farther from the substrate compared to other five residues.


Crystal structure of the γ-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. strainWBC-3, a key enzyme involved in para-Nitrophenol degradation.

Su J, Zhang C, Zhang JJ, Wei T, Zhu D, Zhou NY, Gu Lc - BMC Struct. Biol. (2013)

Substrate binding pocket predicted with the autodock result. A. PnpE is shown in surface model in yellow. NAD is depicted in green sticks and γ-hydroxymuconic semialdehyde is shown in red sticks. B. PnpE is shown in green ribbon model. There are nine amino acids interact with the substrate: C281, E247, F150, I282, F154, H275, F447, W157 and N149.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225490&req=5

Figure 4: Substrate binding pocket predicted with the autodock result. A. PnpE is shown in surface model in yellow. NAD is depicted in green sticks and γ-hydroxymuconic semialdehyde is shown in red sticks. B. PnpE is shown in green ribbon model. There are nine amino acids interact with the substrate: C281, E247, F150, I282, F154, H275, F447, W157 and N149.
Mentions: The substrate γ-hydroxymuconic semialdehyde for PnpE contains an enol structure, which tends to convert into keto under aerobic condition. So it is impracticable to get holo-PnpE structure. To figure out the catalytic mechanism of PnpE without holo-PnpE structure we performed flexible docking for NAD bound PnpE and γ-hydroxymuconic semialdehyde using AUTODOCK (experimental procedure detailed in section 2.7). The docking result is illustrated in Figure 4. The substrate located in a narrow cleft close to NAD. There are nine residues (C281, E247, F150, I282, F154, H275, F447, W157, N149) probably involved in the substrate binding. C281 forms hydrogen bond with the aldehyde group of the substrate directly and may play a key role in the enzymatic reaction. F150, F154, F447, W157, N149, I282 and H275 probably composed a hydrophobic pocket to accommodate the substrate. However, W157 and F447 are a little farther from the substrate compared to other five residues.

Bottom Line: In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde.Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified.The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan 250100, China. n.zhou@pentium.whiov.ac.cn.

ABSTRACT

Background: para-Nitrophenol (PNP) is a highly toxic compound with threats to mammalian health. The pnpE-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in Pseudomonas sp. strain WBC-3, playing a key role in the catabolism of PNP to Krebs cycle intermediates. However, the catalyzing mechanism by PnpE has not been well understood.

Results: Here we report the crystal structures of the apo and NAD bound PnpE. In the PnpE-NAD complex structure, NAD is situated in a cleft of PnpE. The cofactor binding site is composed of two pockets. The adenosine and the first ribose group of NAD bind in one pocket and the nicotinamide ring in the other.

Conclusions: Six amino acids have interactions with the cofactor. They are C281, E247, Q210, W148, I146 and K172. Highly conserved residues C281 and E247 were identified to be critical for its catalytic activity. In addition, flexible docking studies of the enzyme-substrate system were performed to predict the interactions between PnpE and its substrate γ-hydroxymuconic semialdehyde. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the relevant site-directed mutagenesis and their biochemical characterization.

Show MeSH
Related in: MedlinePlus