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

Abstract

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.