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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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Diagram showing substrate preference by NidAB and NidA3B3, indicating that pyrene and fluoranthene were the most effective aromatic substrates, respectively, for these two enzymes. The relative conversion rate for each aromatic substrate is the sum of all metabolites produced, regardless of hydroxylation positions. The rates of pyrene and fluoranthene conversion were set at 100% for NidA and NidA3, respectively, and other substrates were calculated relative to the rates of pyrene and fluoranthene conversion. Biotransformation assays were conducted at least two times, and only representative results are shown as relative conversion rates. The chemical structures and compound names are shown in Fig. 3.
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f2: Diagram showing substrate preference by NidAB and NidA3B3, indicating that pyrene and fluoranthene were the most effective aromatic substrates, respectively, for these two enzymes. The relative conversion rate for each aromatic substrate is the sum of all metabolites produced, regardless of hydroxylation positions. The rates of pyrene and fluoranthene conversion were set at 100% for NidA and NidA3, respectively, and other substrates were calculated relative to the rates of pyrene and fluoranthene conversion. Biotransformation assays were conducted at least two times, and only representative results are shown as relative conversion rates. The chemical structures and compound names are shown in Fig. 3.

Mentions: We investigated the substrate specificities of NidAB and NidA3B3 by testing 13 aromatic substrates, toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene, which include eight aromatic compounds previously used as substrates (11). As shown in Fig. 2, the relative product yields of NidAB and NidA3B3 for the test compounds were compared on the basis of the metabolite profiles and high-performance liquid chromatography (HPLC) peak areas. The results indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds with overlapping substrate specificities. However, NidAB and NidA3B3 showed apparent substrate preferences for pyrene (100:57) and fluoranthene (15:100), respectively, with the highest conversion rates and product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity (Fig. 2 and  3).


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)

Diagram showing substrate preference by NidAB and NidA3B3, indicating that pyrene and fluoranthene were the most effective aromatic substrates, respectively, for these two enzymes. The relative conversion rate for each aromatic substrate is the sum of all metabolites produced, regardless of hydroxylation positions. The rates of pyrene and fluoranthene conversion were set at 100% for NidA and NidA3, respectively, and other substrates were calculated relative to the rates of pyrene and fluoranthene conversion. Biotransformation assays were conducted at least two times, and only representative results are shown as relative conversion rates. The chemical structures and compound names are shown in Fig. 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Diagram showing substrate preference by NidAB and NidA3B3, indicating that pyrene and fluoranthene were the most effective aromatic substrates, respectively, for these two enzymes. The relative conversion rate for each aromatic substrate is the sum of all metabolites produced, regardless of hydroxylation positions. The rates of pyrene and fluoranthene conversion were set at 100% for NidA and NidA3, respectively, and other substrates were calculated relative to the rates of pyrene and fluoranthene conversion. Biotransformation assays were conducted at least two times, and only representative results are shown as relative conversion rates. The chemical structures and compound names are shown in Fig. 3.
Mentions: We investigated the substrate specificities of NidAB and NidA3B3 by testing 13 aromatic substrates, toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene, which include eight aromatic compounds previously used as substrates (11). As shown in Fig. 2, the relative product yields of NidAB and NidA3B3 for the test compounds were compared on the basis of the metabolite profiles and high-performance liquid chromatography (HPLC) peak areas. The results indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds with overlapping substrate specificities. However, NidAB and NidA3B3 showed apparent substrate preferences for pyrene (100:57) and fluoranthene (15:100), respectively, with the highest conversion rates and product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity (Fig. 2 and  3).

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