Identification and characterization of a new three-component nicotinic acid hydroxylase NahAB₁ B₂ from Pusillimonas...

Nicotinic acid (NA) is ubiquitous in nature and its microbial degradation mechanisms are diverse. In this study, Pusillimonas sp. strain T2 was found to be capable of utilizing NA as sole carbon and nitrogen sources. This strain could completely degrade 300 mg l^-1 NA within 3·5 h at 30°C and pH 7·0 and one of the degradation intermediate of NA was identified as 6-hydroxynicotinic acid (6HNA). The draft genome sequences of strain T2 were determined to have a total length of 3·3 M bp and 3054 proteins were predicted. The encoding genes of three-component NA hydroxylase (NahAB₁ B₂ ) genes were identified. The nahAB₁ B₂ genes were heterologously expressed in the non-NA-degrading Shinella sp. strain HZN7. The recombinant HZN7-pBBR-nahAB₁ B₂ converted NA into equimolar 6HNA, while the recombinants HZN7-pBBR-nahAB₁ (lacking component B₂ ) and HZN7-pBBR-nahAB₂ (lacking component B₁ ) could not convert NA. Cell-free extracts of HZN7-pBBR-nahAB₁ B₂ exhibited NA hydroxylase activity. After addition of an artificial electron acceptor (such as phenazine methosulphate, PMS), the NA hydroxylase activity was significantly increased. The K_m and V_max values for NA were 65·94 μmol l^-1 and 260·80 ± 5·69 mU mg^-1 , respectively, using PMS as an electron acceptor. This study provides a novel insight into the NA degradation by bacteria.

Nicotinic acid (NA) serves as a model system for the degradation of N-heterocyclic aromatic compounds and the microbial degradation mechanisms are diverse. This is the first time that a three-component hydroxylase has been identified. This study provides a novel insight into the NA degradation by bacteria.