Abstract
Pseudomonas sp. NCIM 5235 is a caffeine-degrading bacterial strain that metabolizes caffeine by sequential demethylation using methylxanthine demethylases. These enzymes belong to the class of two-component Rieske oxygenases and require an oxidoreductase, NdmD, for efficient catalysis. NdmD in Pseudomonas sp. has a unique domain fusion in its N-terminal that is not observed in any other Rieske oxygenase reductases reported so far. In this report, a ~ 1.7 kb ndmD gene from the gDNA of Pseudomonas sp. has been isolated and has been cloned in a pET28a expression vector. Soluble NdmD was over-expressed in Escherichia coli BL21 cells and purified by Ni2+ NTA chromatography. Monomeric molecular mass of the protein was found to be ~ 65 kDa and optimal activity was observed at 35 °C and pH 8.0. It showed broad substrate specificity with highest Kcat/km of 490.8 ± 17.7 towards cytochrome c. To determine the role of N-terminal Rieske domain in its reductase activity, two deletion constructs Δ114NdmD and Δ250NdmD were made. Cytochrome c reductase (ccr) activity of the NdmD constructs and demethylase activity of NdmA in the presence of NdmD constructs showed that there is no significant difference in the catalytic activity of NdmD upon deletion of its N-terminal Rieske domain. However, there might be some functional and evolutionary significance for the fusion of Rieske domain to NdmD and we hypothesize that this domain fusion is an intermediate phase of evolution towards the development of a more efficient enzyme system for xenobiotic degradation.
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Acknowledgements
The authors acknowledge IITM-SAIF and DST FIST facility for iron estimation by ICP-OES, DSC and CD spectroscopy. SR acknowledges UGC for fellowship.
Funding
This study was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India. Award Number: BT/PR11995/BBE/117/4/2014.
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Retnadhas, S., Gummadi, S.N. Identification and characterization of oxidoreductase component (NdmD) of methylxanthine oxygenase system in Pseudomonas sp. NCIM 5235. Appl Microbiol Biotechnol 102, 7913–7926 (2018). https://doi.org/10.1007/s00253-018-9224-x
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DOI: https://doi.org/10.1007/s00253-018-9224-x