Cloning expression and functional analysis of nic

Authors: Yao Yang Ting Chen Pengjuan Ma Guangdong Shang Yijun Dai Sheng Yuan

Publish Date: 2010/02/02

Volume: 21, Issue: 4, Pages: 593-602

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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 MoV and cytochrome c domains The recombinant NaDHJA1 can catalyze the hydroxylation of nicotinate and 3cyanopyridine NaDHJA1 protein exhibits 528 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 2Fe2S motif and a type I 2Fe2S motif while the same domain in NaDHKT2440 had only a type II 2Fe2S 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 3cyanopyridine 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 3cyanopyridine The subunits swap results indicate the 2Fe2S motif determines the 3cyanopyridine hydroxylation ability which is evidently different from the previous belief that the Mo motif determines substrate specificityWe thank Isabelle ValletGely 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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