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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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Comparison of the cofactor binding pockets of PnpE and BADH (PDB code: 2wme) [29]in electrostatic surface representations. Red represents negative charge and blue represents positive charge. A. Cofactor binding pocket of PnpE. NAD is shown in sticks. B. Cofactor binding pocket of BADH. NADP is depicted in sticks.
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Figure 3: Comparison of the cofactor binding pockets of PnpE and BADH (PDB code: 2wme) [29]in electrostatic surface representations. Red represents negative charge and blue represents positive charge. A. Cofactor binding pocket of PnpE. NAD is shown in sticks. B. Cofactor binding pocket of BADH. NADP is depicted in sticks.

Mentions: So far, both NAD and NADP have been known to be cofactors of the ALDH family. PnpE specifically uses NAD as cofactor. In order to elucidate the structural basis for the coenzyme specificity of PnpE, we compared structures of PnpE-NAD and NADP bound Betaine Aldehyde Dehydrogenase (BADH) (PDB code: 2wme) from Pseudomonas Aeruginosa[29] (Figure 3). Among NADP dependent proteins with known structures, BADH shares the highest sequence identity of 41% with PnpE. Since the only difference between NAD and NADP is that the later has an extra phosphate group, PnpE and BADH should be able to distinguish them by this point. Both PnpE and BADH contain a two-pocket NAD (P) binding site. The first pocket is close to the molecule surface and accommodates the adenosine moiety of NAD, whereas the second, which is deeply located in the active site, can accommodate the nicotinamide ribose moiety. From the structures we can see clearly that the second pocket of BADH is wider than that of PnpE. This means BADH has extra space to accommodate the phosphate group of NADP whereas PnpE does not. On the other hand, in BADH the cofactor bind pocket surface around phosphate group is highly positive charged to match the negative charge of the phosphate group. This is not the case in PnpE. Therefore, PnpE can only use NAD rather than NADP as its cofactor.


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)

Comparison of the cofactor binding pockets of PnpE and BADH (PDB code: 2wme) [29]in electrostatic surface representations. Red represents negative charge and blue represents positive charge. A. Cofactor binding pocket of PnpE. NAD is shown in sticks. B. Cofactor binding pocket of BADH. NADP is depicted in sticks.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Comparison of the cofactor binding pockets of PnpE and BADH (PDB code: 2wme) [29]in electrostatic surface representations. Red represents negative charge and blue represents positive charge. A. Cofactor binding pocket of PnpE. NAD is shown in sticks. B. Cofactor binding pocket of BADH. NADP is depicted in sticks.
Mentions: So far, both NAD and NADP have been known to be cofactors of the ALDH family. PnpE specifically uses NAD as cofactor. In order to elucidate the structural basis for the coenzyme specificity of PnpE, we compared structures of PnpE-NAD and NADP bound Betaine Aldehyde Dehydrogenase (BADH) (PDB code: 2wme) from Pseudomonas Aeruginosa[29] (Figure 3). Among NADP dependent proteins with known structures, BADH shares the highest sequence identity of 41% with PnpE. Since the only difference between NAD and NADP is that the later has an extra phosphate group, PnpE and BADH should be able to distinguish them by this point. Both PnpE and BADH contain a two-pocket NAD (P) binding site. The first pocket is close to the molecule surface and accommodates the adenosine moiety of NAD, whereas the second, which is deeply located in the active site, can accommodate the nicotinamide ribose moiety. From the structures we can see clearly that the second pocket of BADH is wider than that of PnpE. This means BADH has extra space to accommodate the phosphate group of NADP whereas PnpE does not. On the other hand, in BADH the cofactor bind pocket surface around phosphate group is highly positive charged to match the negative charge of the phosphate group. This is not the case in PnpE. Therefore, PnpE can only use NAD rather than NADP as its cofactor.

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