BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Characterization of catabolic meta-nitrophenol nitroreductase from Cupriavidus necator JMP134 Ying Yin & Yi Xiao & Hai-Zhou Liu & Fuhua Hao & Simon Rayner & Huiru Tang & Ning-Yi Zhou Received: 1 February 2010 / Revised: 29 April 2010 / Accepted: 6 May 2010 / Published online: 28 May 2010 # Springer-Verlag 2010 Abstract Cupriavidus necator JMP134 utilizes meta- nitrophenol (MNP) as a sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degra- dation 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 nitro- reductase involved in the initial reaction of MNP degra- dation. 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 K m and high k cat of MnpA as well as MNP-inducible transcription of mnpA suggested that MNP was the physiological substrate for this nitro- reductase. In addition, the phylogenetic analysis revealed that nitroreductases of known physiological function including MnpA constituted a new clade in the nitro- FMN-reductase superfamily. Keywords Catabolism . Cupriavidus necator JMP134 . meta-Nitrophenol . Nitroreductase Introduction Interest in the detoxification of nitrophenols-contaminated environment via bioremediation has lead to the isolation of various microorganisms capable of utilizing nitrophenol isomers as the sole source of carbon, nitrogen, and energy (Spain et al. 1979; Zeyer and Kearney 1984; Bruhn et al. 1987). The initial reactions during the degradation of ortho- nitrophenol (ONP) (Zeyer and Kocher 1988; Xiao et al. 2007) and para-nitrophenol (PNP) (Jain et al. 1994; Zhang et al. 2009) were catalyzed by their corresponding mono- oxygenases. The catabolic pathways, both at molecular and biochemical levels, have been well described for the degradations of ONP (Zeyer and Kocher 1988; Xiao et al. 2007) and PNP through either 1,2,4-benzenetriol (Kitagawa et al. 2004) or hydroquinone (Zhang et al. 2009). In contrast, the reaction of meta-nitrophenol (MNP) metabolism was initiated by an NADPH-dependent partial reduction of the nitro group to yield meta-hydroxylaminophenol (m-HAP) for two alternative pathways, which were proposed via 1,2,4- benzenetriol in Pseudomonas putida B2 (Meulenberg et al. 1996) and via aminohydroquinone in Cupriavidus necator (formerly Ralstonia eutropha) JMP134 (Schenzle et al. 1997). Up to now, no genetic determinates for MNP degradation have been functionally identified, and no nitro- reductase responsible for MNP initial reduction has been characterized for either of the two pathways. Attempts to purify MNP nitroreductase from strain B2 were unsuccessful Y. Yin : Y. Xiao : H.-Z. Liu : S. Rayner : N.-Y. Zhou (*) State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China e-mail: n.zhou@pentium.whiov.ac.cn F. Hao : H. Tang 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 Appl Microbiol Biotechnol (2010) 87:2077–2085 DOI 10.1007/s00253-010-2666-4