A Bph-like nitroarene dioxygenase catalyzes the conversion of 3-nitrotoluene to 3-methylcatechol from Rhodococcus sp. strain ZWL3NT

All reported nitroarene dioxygenases so far are 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 multi-component 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 chemical industry. Microbial degradation of nitroarenes have attracted extensive attentions, not only because this class xenobiotics compounds are recalcitrant in the environment, but also the microbiologists working in this field are curious about the evolutionary origin and process of the nitroarenes 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 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 benzene ring to produce 3-metheylcatechol exclusively from 3NT is also a unique property among the studied nitroarene dioxygenases. This will enrich our understanding of the diversity and origin of nitroarene dioxygenase in microorganisms.