Characterization of Pseudooxynicotine Amine Oxidase of Pseudomonas putida S16 that Is Crucial for Nicotine Degradation

Abstract

Pseudooxynicotine amine oxidase (Pnao) is essential to the pyrrolidine pathway of nicotine degradation of Pseudomonas putida strain S16, which is significant for the detoxification of nicotine, through removing the CH3NH2 group. However, little is known about biochemical mechanism of this enzyme. Here, we characterized its properties and biochemical mechanism. Isotope labeling experiments provided direct evidence that the newly introduced oxygen atom in 3-succinoylsemialdehyde-pyridine is derived from H2O, but not from O2. Pnao was very stable at temperatures below 50 °C; below this temperature, the enzyme activity increased as temperature rose. Site-directed mutagenesis studies showed that residue 180 is important for its thermal stability. In addition, tungstate may enhance the enzyme activity, which has rarely been reported before. Our findings make a further understanding of the crucial Pnao in nicotine degradation.

Figure 1. Biochemical characterization by enzymatic assays of Pnao.

(A) Spectrum curve of Pnao on gel filtration. The column (superdex 200) was marked by a standard protein (Ovalbumin, 48.1 kDa). (B) Kinetic curve of Pnao at 30 °C. The apparent K_m, kcat and k_cat/K_m values for PN at 30 °C are 0.073 ± 0.018 mM, 0.790 ± 0.074 s⁻¹, 10.822 L mol⁻¹ s⁻¹, respectively. (C) Effect of pH on Pnao activity (with immediate detection). (D) Effect of pH on Pnao stability (with overnight incubation). The enzyme was incubated in buffers overnight. (E,F) Effects of metal salts on Pnao activity (with immediate detection) and Pnao stability (with overnight incubation). Metal salts: NaCl, NiCl₂, BaCl₂, CoCl₂, CaCl₂, CuCl₂, MnCl₂, ZnCl₂, KCl, Na₂MoO₄, Na₂WO₄, CdCl₂, BdCl₂, FeCl₃ and MgCl₂. CK, without metal salts. The final concentration of metal salts was 2 mM.

Figure 2. Pnao characteristics by circular dichroism spectrometer (CDS).

(A) Spectrum of wild-type Pnao on CDS analysis. The curve started to change at temperature above 50 °C. (B) The spectrum of mutant Glu180Gly on CDS analysis. The curve started to change at temperature above 25 °C.

Figure 3. SDS-PAGE analysis of site-directed mutagenesis proteins and FAD content detection of Pnao.

(A) SDS-PAGE analysis of purified wildtype Pnao, PnaoE131G, PnaoP149S, PnaoE159G, PnaoE162G, PnaoE174G and PnaoP180S. M, marker protein; 1, wildtype Pnao; 2, purified PnaoE131G; 3, purified PnaoP149S; 4, purified PnaoE159G; 5, purified PnaoE162G; 6 purified PnaoE174G; and 7, purified PnaoP180S. (B) FAD content of wildtype, mutants and Pnao with different metal salts. C, control group; W, tungsten; Mo, molybdenum; Fe, ferric iron.

Figure 4. Mechanism of Pnao reaction as determined by LC-MS analysis.

(A) The control group. A molecular peak was observed at m/z 164, which is the same as molecular weight of SAP. (B) The ¹⁸O₂ group. A molecular peak was also observed at m/z 164, which means that O₂ is not the source of the newly introduced oxygen atom. (C) The H₂¹⁸O group. A molecular peak was observed at m/z 166. Thus we inferred that the newly introduced oxygen atom of succinate is derived from H₂O not from O₂. (D) A general view of the reaction process of Pnao. In the first step, PN is activated via the reduction of FAD and a carbon-nitrogen double bond is formed. Then, H₂O attaches the carbon-nitrogen double bond and a hypothetic, extremely unstable intermediate is generated. This intermediate is transformed to SAP and CH₃NH₂ quickly. Finally, FAD is regenerated via the oxidation to make preparation for the next reaction.