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 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.