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Characterization of catabolic meta-nitrophenol nitroreductase from Cupriavidus necator JMP134

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Abstract

Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as a sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that the mnp cluster might have played important roles in MNP degradation. In order to experimentally confirm the prediction, we have now characterized mnpA-encoded meta-nitrophenol nitroreductase involved in the initial reaction of MNP degradation. Real-time PCR analysis indicated that mnpA played an essential role in MNP degradation. MnpA was purified to homogeneity as His-tagged proteins and was considered to be a dimer as determined by gel filtration. MnpA was an MNP nitroreductase with a tightly bound flavin mononucleotide (FMN), catalyzing the partial reduction of MNP to meta-hydroxylaminophenol via meta-nitrosophenol in the presence of NADPH and oxygen. The accumulation of meta-nitrosophenol was confirmed with the results of liquid chromatography–diode array detection and time-of-flight mass spectrometry for the first time. The low Km and high kcat of MnpA as well as MNP-inducible transcription of mnpA suggested that MNP was the physiological substrate for this nitroreductase. In addition, the phylogenetic analysis revealed that nitroreductases of known physiological function including MnpA constituted a new clade in the nitro-FMN-reductase superfamily.

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Acknowledgments

We acknowledge the financial supports from the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-072-3) and National Natural Science Foundation of China (30730002 and 20825520).

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Authors and Affiliations

  1. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China

    Ying Yin, Yi Xiao, Hai-Zhou Liu, Simon Rayner & Ning-Yi Zhou

  2. State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, China

    Fuhua Hao & Huiru Tang

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  1. Ying Yin
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  2. Yi Xiao
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  3. Hai-Zhou Liu
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  4. Fuhua Hao
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  6. Huiru Tang
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  7. Ning-Yi Zhou
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Correspondence to Ning-Yi Zhou.

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Yin, Y., Xiao, Y., Liu, HZ. et al. Characterization of catabolic meta-nitrophenol nitroreductase from Cupriavidus necator JMP134. Appl Microbiol Biotechnol 87, 2077–2085 (2010). https://doi.org/10.1007/s00253-010-2666-4

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