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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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Enzyme activities of wild type PnpE and different mutants. The activity of the wild type is set to 100%. C281A, E247K, N149D, F150A, H275E, I282D, W157A, F154A, F447A are mutants made according to docking results.
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Figure 5: Enzyme activities of wild type PnpE and different mutants. The activity of the wild type is set to 100%. C281A, E247K, N149D, F150A, H275E, I282D, W157A, F154A, F447A are mutants made according to docking results.

Mentions: To confirm the molecular docking result, nine single amino acid mutants (C281A, E247K, N149D, F150A, F154A, W157A, H275E, F447A, I282D) were generated using a two-step PCR strategy. Their enzyme activities were then measured and repeated four times (Figure 5). The results showed the mutants of C281A, E247K, N149D, F150A, H275E and I282D completely lost their activity. The activity of F154A decreased about 60%. The activity of W157Aand F447A decreased about 20%. C281 and E247, which are highly conserved across the ALDH family members, have been known to play essential roles in the catalytic process. The loss of PnpE activity by N149D, F150A, H275E, I282D, and the significant decrease of the activity by F154A also argue that these amino acids play important roles in the substrate binding. W157 and F447 do not contact substrate directly, thus W157A and F447A only affected enzymatic activity modestly, compared to the significant effects of other seven mutations. These data are consistent with the result of AUTODOCK calculations.


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)

Enzyme activities of wild type PnpE and different mutants. The activity of the wild type is set to 100%. C281A, E247K, N149D, F150A, H275E, I282D, W157A, F154A, F447A are mutants made according to docking results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Enzyme activities of wild type PnpE and different mutants. The activity of the wild type is set to 100%. C281A, E247K, N149D, F150A, H275E, I282D, W157A, F154A, F447A are mutants made according to docking results.
Mentions: To confirm the molecular docking result, nine single amino acid mutants (C281A, E247K, N149D, F150A, F154A, W157A, H275E, F447A, I282D) were generated using a two-step PCR strategy. Their enzyme activities were then measured and repeated four times (Figure 5). The results showed the mutants of C281A, E247K, N149D, F150A, H275E and I282D completely lost their activity. The activity of F154A decreased about 60%. The activity of W157Aand F447A decreased about 20%. C281 and E247, which are highly conserved across the ALDH family members, have been known to play essential roles in the catalytic process. The loss of PnpE activity by N149D, F150A, H275E, I282D, and the significant decrease of the activity by F154A also argue that these amino acids play important roles in the substrate binding. W157 and F447 do not contact substrate directly, thus W157A and F447A only affected enzymatic activity modestly, compared to the significant effects of other seven mutations. These data are consistent with the result of AUTODOCK calculations.

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