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Substrate specificity and structural characteristics of the novel Rieske nonheme iron aromatic ring-hydroxylating oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1.

Kweon O, Kim SJ, Freeman JP, Song J, Baek S, Cerniglia CE - MBio (2010)

Bottom Line: Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase.Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs.Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA.

ABSTRACT
The Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 have been implicated in the initial oxidation of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), forming cis-dihydrodiols. To clarify how these two RHOs are functionally different with respect to the degradation of HMW PAHs, we investigated their substrate specificities to 13 representative aromatic substrates (toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene) by enzyme reconstitution studies of Escherichia coli. Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase. Metabolite profiles indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds, producing various isomeric dihydrodiol and phenolic compounds. NidAB and NidA3B3 showed the highest conversion rates for pyrene and fluoranthene, respectively, with high product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity. Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs. The NidAB and NidA3B3 systems showed the largest substrate-binding pockets in the active sites, which satisfies spatial requirements for accepting HMW PAHs. Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons.

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Substrate ranges of NidAB, NidA3B3 from M. vanbaalenii PYR-1, CARDO from Pseudomonas sp. strain CA10, and PhnI from Sphingomonas sp. strain CHY-1. The relative conversion rate (as a percentage) for the test aromatic compounds with respect to the most favorable substrate (100%) was plotted.
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f4: Substrate ranges of NidAB, NidA3B3 from M. vanbaalenii PYR-1, CARDO from Pseudomonas sp. strain CA10, and PhnI from Sphingomonas sp. strain CHY-1. The relative conversion rate (as a percentage) for the test aromatic compounds with respect to the most favorable substrate (100%) was plotted.

Mentions: To address the relationship between substrate specificity and active site, we compared the Nid systems to other well-organized RHO information. As shown in Fig. 4, the substrate specificities of NidAB and NidA3B3 are different from those of other RHO enzymes. Pyrene and fluoranthene were the best substrates for NidAB and NidA3B3, respectively, whereas naphthalene and biphenyl were the most preferred substrates for PhnI from Sphingomonas sp. strain CHY-1 and carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp. strain CA10 (29), respectively. As shown in Table 2, the amino acid sequence identities and root mean square deviation (RMSD) (Cα) of the Nid systems indicate their structural differences from other RHOs. In particular, the angular dioxygenase CARDO from Janthinobacterium sp. strain J3 showed the lowest structural similarity to the Nid systems. Table 3 and Fig. 5 show comparisons of the active sites, in which the volume of the active site corresponds to the area defined by the van der Waals surface of the residues that contribute to the overall topology of the active sites. The dimensions of the active sites were also measured to correlate them with the active-site volume. NidA and NidA3 showed the largest active sites in both volume (509 and 613 Å3, respectively) and size (14.5 Å by 7.4 Å by 14.8 Å and 15.8 Å by 10.0 Å by 17.8 Å [width, height, and length, respectively]). The presence of aromatic amino acids in the substrate-binding pockets is a common feature of many RHOs. The structural overlays of several RHO active sites on that of NDO derived from Pseudomonas putida NCIB 9816-4 and bound to phenanthrene revealed that the aromatic amino acids corresponding to Phe-202, Phe-352, and Trp-358 of the NDO are spatially conserved in the 3-D structure (Fig. 6a). In particular, two Phe residues, at positions 202 and 352 of the NDO, were perfectly conserved in these RHOs. The conserved aromatic amino acids in the active sites of NidA and NidA3 are Phe-193, Phe-347, and Phe-353 and Phe-193, Phe-345, and Phe-351, respectively. In the analysis of interaction between RHO active sites and their substrates, these conserved aromatic amino acids were revealed to be involved in the aromatic interactions (Fig. 6b, c, and d; see also Table SA3 in the supplemental material). Aromatic interactions in combination with hydrophobic interactions could play a role in substrate binding (see Fig. SA2 in the supplemental material). Residues of NidA and NidA3 in contact with the bound pyrene and fluoranthene, respectively, which are likely to be involved in aromatic and hydrophobic interactions, were proposed (Fig. SA2).


Substrate specificity and structural characteristics of the novel Rieske nonheme iron aromatic ring-hydroxylating oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1.

Kweon O, Kim SJ, Freeman JP, Song J, Baek S, Cerniglia CE - MBio (2010)

Substrate ranges of NidAB, NidA3B3 from M. vanbaalenii PYR-1, CARDO from Pseudomonas sp. strain CA10, and PhnI from Sphingomonas sp. strain CHY-1. The relative conversion rate (as a percentage) for the test aromatic compounds with respect to the most favorable substrate (100%) was plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921158&req=5

f4: Substrate ranges of NidAB, NidA3B3 from M. vanbaalenii PYR-1, CARDO from Pseudomonas sp. strain CA10, and PhnI from Sphingomonas sp. strain CHY-1. The relative conversion rate (as a percentage) for the test aromatic compounds with respect to the most favorable substrate (100%) was plotted.
Mentions: To address the relationship between substrate specificity and active site, we compared the Nid systems to other well-organized RHO information. As shown in Fig. 4, the substrate specificities of NidAB and NidA3B3 are different from those of other RHO enzymes. Pyrene and fluoranthene were the best substrates for NidAB and NidA3B3, respectively, whereas naphthalene and biphenyl were the most preferred substrates for PhnI from Sphingomonas sp. strain CHY-1 and carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp. strain CA10 (29), respectively. As shown in Table 2, the amino acid sequence identities and root mean square deviation (RMSD) (Cα) of the Nid systems indicate their structural differences from other RHOs. In particular, the angular dioxygenase CARDO from Janthinobacterium sp. strain J3 showed the lowest structural similarity to the Nid systems. Table 3 and Fig. 5 show comparisons of the active sites, in which the volume of the active site corresponds to the area defined by the van der Waals surface of the residues that contribute to the overall topology of the active sites. The dimensions of the active sites were also measured to correlate them with the active-site volume. NidA and NidA3 showed the largest active sites in both volume (509 and 613 Å3, respectively) and size (14.5 Å by 7.4 Å by 14.8 Å and 15.8 Å by 10.0 Å by 17.8 Å [width, height, and length, respectively]). The presence of aromatic amino acids in the substrate-binding pockets is a common feature of many RHOs. The structural overlays of several RHO active sites on that of NDO derived from Pseudomonas putida NCIB 9816-4 and bound to phenanthrene revealed that the aromatic amino acids corresponding to Phe-202, Phe-352, and Trp-358 of the NDO are spatially conserved in the 3-D structure (Fig. 6a). In particular, two Phe residues, at positions 202 and 352 of the NDO, were perfectly conserved in these RHOs. The conserved aromatic amino acids in the active sites of NidA and NidA3 are Phe-193, Phe-347, and Phe-353 and Phe-193, Phe-345, and Phe-351, respectively. In the analysis of interaction between RHO active sites and their substrates, these conserved aromatic amino acids were revealed to be involved in the aromatic interactions (Fig. 6b, c, and d; see also Table SA3 in the supplemental material). Aromatic interactions in combination with hydrophobic interactions could play a role in substrate binding (see Fig. SA2 in the supplemental material). Residues of NidA and NidA3 in contact with the bound pyrene and fluoranthene, respectively, which are likely to be involved in aromatic and hydrophobic interactions, were proposed (Fig. SA2).

Bottom Line: Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase.Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs.Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas, USA.

ABSTRACT
The Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 have been implicated in the initial oxidation of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), forming cis-dihydrodiols. To clarify how these two RHOs are functionally different with respect to the degradation of HMW PAHs, we investigated their substrate specificities to 13 representative aromatic substrates (toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene) by enzyme reconstitution studies of Escherichia coli. Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase. Metabolite profiles indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds, producing various isomeric dihydrodiol and phenolic compounds. NidAB and NidA3B3 showed the highest conversion rates for pyrene and fluoranthene, respectively, with high product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity. Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs. The NidAB and NidA3B3 systems showed the largest substrate-binding pockets in the active sites, which satisfies spatial requirements for accepting HMW PAHs. Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons.

Show MeSH
Related in: MedlinePlus