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 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.

Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases

“…Rieske oxygenases in particular are underexplored in this regard, with only twelve available structures of different enzymes in the Protein Data Bank 21 . Five of these enzymes, including biphenyl dioxygenase [22][23][24][25] , 3nitrotoluene dioxygenase 26 , naphthalene 1,2-dioxygenase [27][28][29][30] , cumene dioxygenase 31 , and nitrobenzene dioxygenase 32 , are clustered together in the sequence similarity network (SSN) presented in Fig. 1a (light blue) and catalyze the oxidative degradation of aromatic compounds (IPR001663, IPR015879).…”

A Nag‐like dioxygenase initiates 3,4‐dichloronitrobenzene degradation via 4,5‐dichlorocatechol in Diaphorobacter sp. strain JS3050

“…strain U2 (Zhou et al ., 2002). It has a gene arrangement and biochemical motifs ([2Fe‐2S] cluster binding site) similar to the reported group III of Rieske non‐heme iron dioxygenases (Parales and Resnick, 2006) that catalyses dioxygenation of 2,4‐dinitrotoluene (Suen and Spain, 1993), nitrobenzene (Nishino and Spain, 1995), 2‐nitrotoluene (An et al ., 1994), 3‐nitrotoluene (Kumari et al ., 2017), 2‐chloronitrobenzene (Liu et al ., 2011) and naphthalene (Zhou et al ., 2001). Thus the cluster of genes would be expected to encode synthesis of a functional nitroarene dioxygenase system that would catalyse conversion of 3,4‐DCNB to a dichlorocatechol.…”

“…The α subunit of 3‐NT dioxygenase from Diaphorobacter sp. strain DS2 (pdb number: 5xbp), with an 87% amino acid sequence identity with the alpha subunit of 3,4‐DCNB dioxygenase, was used as the modelling template (Kumari et al ., 2017). The GMQE and QMEAN scores of 0.98 and −1.17 respectively showed the reliability of this model.…”

A Recently Assembled Degradation Pathway for 2,3-Dichloronitrobenzene in Diaphorobacter sp. Strain JS3051

“…The clade II nitroarene dioxygenases accept 2/3-substituted nitroarenes, whereas the clade I nitroarene dioxygenases prefer 3,4-substituted substrates ( Fig. 4A and B ) ( 21 , 31 , 38 – 40 ). The position of the substituents seems to have more influence than the type of the groups on the substrate preferences of nitroarene dioxygenases.…”