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Cloning, expression and functional analysis of nicotinate dehydrogenase gene cluster from Comamonas testosteroni JA1 that can hydroxylate 3-cyanopyridine

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Abstract

A nicotinate dehydrogenase (NaDH) gene cluster was cloned from Comamonas testosteroni JA1. The enzyme, termed NaDHJA1, is composed of 21, 82, and 46 kDa subunits, respectivley containing [2Fe2S], Mo(V) and cytochrome c domains. The recombinant NaDHJA1 can catalyze the hydroxylation of nicotinate and 3-cyanopyridine. NaDHJA1 protein exhibits 52.8% identity to the amino acid sequence of NaDHKT2440 from P. putida KT2440. Sequence alignment analysis showed that the [2Fe2S] domain in NaDHJA1 had a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, while the same domain in NaDHKT2440 had only a type II [2Fe-2S] motif. NaDHKT2440 had an additional hypoxanthine dehydrogenase motif that NaDHJA1 does not have. When the small unit of NaDHJA1 was replaced by the small subunit from NaDHKT2440, the hybrid protein was able to catalyze the hydroxylation of nicotinate, but lost the ability to catalyze hydroxylation of 3-cyanopyridine. In contrast, after replacement of the small subunit of NaDHKT2440 with the small subunit from NaDHJA1, the resulting hybrid protein NaDHJAS+KTL acquired the ability to hydroxylate 3-cyanopyridine. The subunits swap results indicate the [2Fe2S] motif determines the 3-cyanopyridine hydroxylation ability, which is evidently different from the previous belief that the Mo motif determines substrate specificity.

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Acknowledgments

We thank Isabelle Vallet-Gely, Bruno Lemaitre laboratory, FRANCE, and He Jian, Nanjing Agriculture University, Department of Life Science, for kindly providing Pseudomonas putida L48 and Pseudomonas putida KT2440, respectively. This work was supported by the Key Fundamental Research Program of Jiangsu Higher Education Institution of China (06KJA21016), the Natural Science Foundation of Jiangsu Higher Education Institution of China (04KJB180071).

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  1. Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Science, Nanjing Normal University, 1Wenyuan Rd, Nanjing, 210046, People’s Rebuplic of China

    Yao Yang, Ting Chen, Pengjuan Ma, Guangdong Shang, Yijun Dai & Sheng Yuan

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  1. Yao Yang
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  2. Ting Chen
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Correspondence to Sheng Yuan.

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Yang, Y., Chen, T., Ma, P. et al. Cloning, expression and functional analysis of nicotinate dehydrogenase gene cluster from Comamonas testosteroni JA1 that can hydroxylate 3-cyanopyridine. Biodegradation 21, 593–602 (2010). https://doi.org/10.1007/s10532-010-9327-2

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