Cloning, expression and functional analysis of nicotinate dehydrogenase gene cluster from Comamonas testosteroni JA1 that can hydroxylate 3-cyanopyridine.
A nicotinate dehydrogenase (NaDH) gene cluster was cloned from Comamonas testosteroni JA1. The enzyme, termed NaDH(JA1), is composed of 21, 82, and 46 kDa subunits, respectivley containing [2Fe2S], Mo(V) and cytochrome c domains. The recombinant NaDH(JA1) can catalyze the hydroxylation of nicotinate and 3-cyanopyridine. NaDH(JA1) protein exhibits 52.8% identity to the amino acid sequence of NaDH(KT2440) from P. putida KT2440. Sequence alignment analysis showed that the [2Fe2S] domain in NaDH(JA1) had a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, while the same domain in NaDH(KT2440) had only a type II [2Fe-2S] motif. NaDH(KT2440) had an additional hypoxanthine dehydrogenase motif that NaDH(JA1) does not have. When the small unit of NaDH(JA1) was replaced by the small subunit from NaDH(KT2440), 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 NaDH(KT2440) with the small subunit from NaDH(JA1), the resulting hybrid protein NaDH(JAS+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.
Yang
Yao
Y
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.
Chen
Ting
T
Ma
Pengjuan
P
Shang
Guangdong
G
Dai
Yijun
Y
Yuan
Sheng
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D020604:Comamonas testosteroni / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006900:Hydroxylation; D008969:Molecular Sequence Data; D005810:Multigene Family; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011725:Pyridines; C021862:nicotinate dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; C020729:3-cyanopyridine