Dataset Information

Structural and functional studies of ferredoxin and oxygenase components of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.

ABSTRACT: 3-nitrotoluene dioxygenase (3NTDO) from Diaphorobacter sp. strain DS2 catalyses the conversion of 3-nitrotoluene (3NT) into a mixture of 3- and 4-methylcatechols with release of nitrite. We report here, X-ray crystal structures of oxygenase and ferredoxin components of 3NTDO at 2.9 Å and 2.4 Å, respectively. The residues responsible for nitrite release in 3NTDO were further probed by four single and two double mutations in the catalytic site of ?-subunit of the dioxygenase. Modification of Val 350 to Phe, Ile 204 to Ala, and Asn258 to Val by site directed mutagenesis resulted in inactive enzymes revealing the importance of these residues in catalysis. Docking studies of meta nitrotoluene to the active site of 3NTDO suggested possible orientations of binding that favor the formation of 3-methylcatechol (3MC) over 4-methylcatechol energetically. The electron transfer pathway from ferredoxin subunit to the active site of the oxygenase subunit is also proposed.

SUBMITTER: Kumari A

PROVIDER: S-EPMC5407579 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structural and functional studies of ferredoxin and oxygenase components of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.

Kumari Archana A Singh Deepak D Ramaswamy S S Ramanathan Gurunath G

PloS one 20170427 4

3-nitrotoluene dioxygenase (3NTDO) from Diaphorobacter sp. strain DS2 catalyses the conversion of 3-nitrotoluene (3NT) into a mixture of 3- and 4-methylcatechols with release of nitrite. We report here, X-ray crystal structures of oxygenase and ferredoxin components of 3NTDO at 2.9 Å and 2.4 Å, respectively. The residues responsible for nitrite release in 3NTDO were further probed by four single and two double mutations in the catalytic site of α-subunit of the dioxygenase. Modification of Val 3 ...[more]

PMID: 28448625

Similar Datasets

Project description:BackgroundThe initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-FB1). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-OB1), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo[a]pyrene.ResultsIn this study, crystal structures of BPDO-OB1 in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-FB1 has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted.ConclusionThis is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron-sulfur cluster. Because this ferredoxin is used by multiple oxygenases present in the B1 organism, this ferredoxin-oxygenase system provides the structural platform to dissect the balance between promiscuity and selectivity in protein-protein electron transport systems.

Project description:All nitroarene dioxygenases reported so far originated from Nag-like naphthalene dioxygenase of Gram-negative strains, belonging to group III of aromatic ring-hydroxylating oxygenases (RHOs). Gram-positive Rhodococcus sp. strain ZWL3NT utilizes 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. It was also reported that 3NT degradation was constitutive and the intermediate was 3-methylcatechol. In this study, a gene cluster (bndA1A2A3A4) encoding a multicomponent dioxygenase, belonging to group IV of RHOs, was identified. Recombinant Rhodococcus imtechensis RKJ300 carrying bndA1A2A3A4 exhibited 3NT dioxygenase activity, converting 3NT into 3-methylcatechol exclusively, with nitrite release. The identity of the product 3-methylcatechol was confirmed using liquid chromatography-mass spectrometry. A time course of biotransformation showed that the 3NT consumption was almost equal to the 3-methylcatechol accumulation, indicating a stoichiometry conversion of 3NT to 3-methylcatechol. Unlike reported Nag-like dioxygenases transforming 3NT into 4-methylcatechol or both 4-methylcatechol and 3-methylcatechol, this Bph-like dioxygenase (dioxygenases homologous to the biphenyl dioxygenase from Rhodococcus sp. strain RHA1) converts 3NT to 3-methylcatechol without forming 4-methylcatechol. Furthermore, whole-cell biotransformation of strain RKJ300 with bndA1A2A3A4 and strain ZWL3NT exhibited the extended and same substrate specificity against a number of nitrobenzene or substituted nitrobenzenes, suggesting that BndA1A2A3A4 is likely the native form of 3NT dioxygenase in strain ZWL3NT.IMPORTANCE Nitroarenes are synthetic molecules widely used in the chemical industry. Microbial degradation of nitroarenes has attracted extensive attention, not only because this class of xenobiotic compounds is recalcitrant in the environment but also because the microbiologists working in this field are curious about the evolutionary origin and process of the nitroarene dioxygenases catalyzing the initial reaction in the catabolism. In contrast to previously reported nitroarene dioxygenases from Gram-negative strains, which originated from a Nag-like naphthalene dioxygenase, the 3-nitrotoluene (3NT) dioxygenase in this study is from a Gram-positive strain and is an example of a Bph-like nitroarene dioxygenase. The preference of hydroxylation of this enzyme at the 2,3 positions of the benzene ring to produce 3-methylcatechol exclusively from 3NT is also a unique property among the studied nitroarene dioxygenases. These findings will enrich our understanding of the diversity and origin of nitroarene dioxygenase in microorganisms.

Dataset Information

Structural and functional studies of ferredoxin and oxygenase components of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.

Publications

Structural and functional studies of ferredoxin and oxygenase components of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets