Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrollidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an ORF adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), that has a typical signal peptide of bacterial Paz. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that, with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol min-1 mg protein-1 , where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2 O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)+ , O2 , and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria. IMPORTANCE Nicotine is a toxic and addictive N -heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become the major concerns of health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [lsqb]2Fe2S[rsqb] clusters, and usually one FAD as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge in microbial degradation of N -heterocyclic aromatic compound.