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

Archana Kumari,S Ramaswamy,Deepak Singh,Gurunath Ramanathan,Eugene A Permyakov

doi:10.1371/journal.pone.0176398

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 α-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.

Highlights

Nitroaromatic compounds are widely used in the production of dyes, pigments, pesticides and explosives [1]
Cells were harvested by centrifugation and pDS21 cells were resuspended in BTGD buffer [50mM bis-tris, 5% (v/v) glycerol, 1mM dithiothreitol] and pDS5 and pDS2 cells were suspended in MGD buffer [25mM MOPS, 5% glycerol, 1mM DTT] with 50 and 100mM NaCl, respectively and frozen at -70 ̊C
A structure of ferredoxin (3NTDO-FDS2) (Fig 1A) was solved to 2.4Å resolution using the molecular replacement method and by using the ferredoxin component of Naphthalene dioxygenase (NDO) (NDO-F9816-4) (PDB code: 2QPZ)[27] from Pseudomonas putida NCIB9816-4 as a search model

Summary

Introduction

Nitroaromatic compounds are widely used in the production of dyes, pigments, pesticides and explosives [1]. We report the crystal structure of the ferredoxin component of 3NTDO and we propose the residues involved in the electron transfer pathway from ferredoxin to the mononuclear iron center at the active site of the oxygenase for naphthalene dioxygenase family members. A structure of ferredoxin (3NTDO-FDS2) (Fig 1A) was solved to 2.4Å resolution using the molecular replacement method and by using the ferredoxin component of NDO (NDO-F9816-4) (PDB code: 2QPZ)[27] from Pseudomonas putida NCIB9816-4 as a search model.

Results

Conclusion