Identification and Characterization of Cotinine Hydroxylase CotA that Initiates biodegradation of Wastewater Micropollutant Cotinine in Nocardioides sp. Strain JQ2195

Cotinine is a stable toxic contaminant, produced as a byproduct of smoking. It is of emerging concern due to its global distribution in aquatic environments. Microorganisms have the potential to degrade cotinine, however, the genetic mechanisms of this process are unknown. Nocardioides sp. strain JQ2195 is a pure culture strain that has been reported to degrade cotinine at micropollutant concentrations. This strain utilizes cotinine as its sole carbon and nitrogen source. In this study, a 50 kb gene cluster (designated as cot ) involved in cotinine degradation, was predicted based on genomic and transcriptomic analyses. A novel three-component cotinine hydroxylase gene (designated as cotA1A2A3 ), which initiated cotinine catabolism was identified and characterized. CotA from Shinella sp. HZN7 was heterologously expressed and purified, and shown to convert cotinine into 6-hydroxycotinine. H 2 18 O-labelling and ESI-MS analysis confirmed that the hydroxyl group incorporated into 6-hydroxycotinine was derived from water. This study provides new molecular insights into the microbial metabolism of heterocyclic chemical pollutants. IMPORTANCE In the human body, cotinine is the major metabolite of nicotine, and 10–15% of generated cotinine is excreted in urine. Cotinine is a structural analogue of nicotine and is much more stable than nicotine. Increased tobacco consumption has led to high environmental concentrations of cotinine, which may have detrimental effects on aquatic ecosystems and human health. Nocardioides sp. strain JQ2195 is a unique cotinine-degrading bacterium. However, the underlying genetic and biochemical foundations of cotinine degradation are still unknown. In this study, a 50 kb gene cluster (designated cot ) was identified by genomic and transcriptomic analyses as being involved in the degradation of cotinine. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3 ) catalyzed cotinine to 6-hydroxy-cotinine. This study provides new molecular insights into the microbial degradation and enzymatic transformation of cotinine.