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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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The oxidation process of γ-hydroxymuconic semialdehyde to maleyacetate with NAD as cofactor by PnpE.
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Figure 7: The oxidation process of γ-hydroxymuconic semialdehyde to maleyacetate with NAD as cofactor by PnpE.

Mentions: Sequence-based alignment of PnpE and previously determined structures of ALDHs shows that two residues (E247 and C281) are highly conserved across different species (Figure 6). These two amino acids act as the catalytic residues that transport electron, according to the mechanism elucidated in other homologous proteins [30-32]. In this regard, PnpE may have a similar catalytic mechanism (Figure 7). The catalytic C281 plays a role as a nucleophilic reagent. It attacks the hydroxymuconic semialdehyde substrate and forms a covalent intermediate. Next, the hydride from this intermediate transfers to NAD to form NADH and the thioacyl enzyme intermediate. The other catalytic residue E247 directly attacks the acyl-sulfur bond and releases the acid product prior to NADH dissociation.


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)

The oxidation process of γ-hydroxymuconic semialdehyde to maleyacetate with NAD as cofactor by PnpE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: The oxidation process of γ-hydroxymuconic semialdehyde to maleyacetate with NAD as cofactor by PnpE.
Mentions: Sequence-based alignment of PnpE and previously determined structures of ALDHs shows that two residues (E247 and C281) are highly conserved across different species (Figure 6). These two amino acids act as the catalytic residues that transport electron, according to the mechanism elucidated in other homologous proteins [30-32]. In this regard, PnpE may have a similar catalytic mechanism (Figure 7). The catalytic C281 plays a role as a nucleophilic reagent. It attacks the hydroxymuconic semialdehyde substrate and forms a covalent intermediate. Next, the hydride from this intermediate transfers to NAD to form NADH and the thioacyl enzyme intermediate. The other catalytic residue E247 directly attacks the acyl-sulfur bond and releases the acid product prior to NADH dissociation.

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