Article Figures & Data
Figures
- FIG 1
Overall structure of the nicotine oxidoreductase (NicA2) from Pseudomonas putida S16 in complex with the cofactor FAD and the substrate nicotine. (A) The upstream pathway of nicotine degradation in Pseudomonas putida S16. NicA2 catalyzes the dehydrogenation of the pyrrolidine moiety of nicotine to yield N-methylmyosmine, which is spontaneously hydrated to produce pseudooxynicotine (PN). PN is further converted to 3-succinoylsemialdehyde-pyridine (SAP) and then to 3-succinylpyridine (SP) by two sequential reactions catalyzed by Pnao and Sapd, respectively. SP is then hydroxylated by the trimeric SP monooxygenase (Spm) to form 6-hydroxy-3-succinylpyridine (HSP). (B) Crystal structure of the NicA2–FAD–nicotine ternary complex. The substrate-binding domain and the FAD-binding domain of NicA2 are color-coded green and yellow, respectively. FAD and nicotine are shown as sticks, color-coded blue and red, respectively. The entrance path for nicotine and the exit path for the reaction product PN are indicated by arrows. (C) The structure in panel B is rotated 90° counterclockwise. Note the kink between helices α3a and α3b, which breaks the continuity of these two helices.
- FIG 2
The exit passage for the reaction product PN within the NicA2 enzyme is occluded by several bulky residues. (A and B) NicA2 buries its substrate nicotine completely inside. NicA2 is shown as a surface representation. The nicotine molecule cannot be seen when viewed from outside the surface of NicA2 when its surface is set at 0% transparency (A) but can be seen to exist inside NicA2 when its surface is set at 40% transparency (B). (C) The exit passage of NicA2 for the reaction product PN is blocked by several bulky residues, including F163, Y214, Y218, Y242, M246, E249, F353, F355, and W364.
- FIG 3
NicA2-catalyzed oxidation of nicotine is complete within seconds, whereas release of the reaction product PN from NicA2 is a much slower process. (A) NicA2-catalyzed oxidation of nicotine was complete within 10 s, as demonstrated by the UV-Vis spectroscopy assay. In the absence of nicotine, purified NicA2 protein exhibited absorbance peaks at 375 nm and 450 nm (blue curve), indicating that its associated FAD cofactor was in the oxidized state. On the other hand, 10 s after the addition of nicotine to NicA2, the absorbance peaks at 375 nm and 450 nm almost totally disappeared (green curve), suggesting that the associated FAD was reduced to FADH2 and the substrate nicotine was oxidized at the same time. The bump at 370 nm is due to the spectrometer’s switching of the running mode from UV scan to visible-light scan. (B) The release of the reaction product PN from NicA2 is a much slower process than the NicA2-catalyzed oxidation of nicotine. NicA2 was mixed with nicotine for 1.5 h, and then the NicA2 protein was removed by passing the mixture through a Ni2+-NTA affinity column. (Left) Gas chromatography was performed for the total mixture (total PN) (top) and the eluted fraction (free PN) (bottom) to measure the amounts of total PN generated in the reaction and PN released into solution from NicA2. (Right) Quantification of total and released PN. Three duplicate samples were set for each group. Error bars represent standard deviations.
- FIG 4
Knockout of the Pnao gene in P. putida S16 cells resulted in hindrance of bacterial cell growth in both nutrient-rich and nutrient-poor media, and mutations of the bulky residues at the PN exit passage of NicA2 to small-side-chain amino acids caused the bacteria to grow much more slowly, a defect that was rescued by the coexpression of Pnao. (A and B) Bacterial growth curves of WT (blue), ΔPnao (magenta), ΔSpm (green), and ΔSapd (red) strains in LB medium (A) or glycerol medium (B). (A) In the nutrient-rich LB medium, knockout of the spm or sapd gene did not have much adverse effect on the cell growth rate, whereas knockout of the Pnao gene caused the cells to grow much more slowly. The growth of WT P. putida S16 cells served as a control. (B) In the nutrient-poor glycerol medium, knockout of the Pnao gene caused the cells to grow much more slowly, while knockout of the Spm or Sapd gene did not have obvious effects. (C) The growth of a P. putida S16 cell culture transformed with the empty pME6032 plasmid was not much affected by the addition of IPTG and nicotine. (D) There was a lag time of 2 h for the growth of P. putida S16 cells overexpressing WT NicA2 upon the addition of IPTG and nicotine, but the cells could still grow up to an OD600 of ∼1.0 after 12 h of culturing. (E) The lag time for P. putida S16 cells overexpressing the 9AA mutant of NicA2 upon the addition of IPTG and nicotine was 4 h, twice as long as that for WT NicA2. Furthermore, the cell density (OD600) after 12 h of growth remained below 0.8. (F) When P. putida S16 cells were transformed with a pME6032 plasmid coexpressing both the 9AA mutant of NicA2 and the downstream enzyme Pnao, the addition of IPTG and nicotine did not affect the growth rate of bacterial cells. All experiments were performed in duplicate. (G) Overexpression of the 9AA mutant of NicA2 caused PN to accumulate, while coexpression of Pnao with the 9AA mutant of NicA2 prevented the accumulation. Shown are amounts of PN at different time points in bacterial cultures of P. putida S16 transformed with either the empty pME6032 plasmid, WT NicA2, the 9AA mutant of NicA2, or the 9AA mutant of NicA2 together with Pnao, as measured by gas chromatography. Three duplicate samples were set for each group. Error bars represent standard deviations.
Tables
- TABLE 1
Data collection and refinement statistics
↵a Rmerge = ΣhΣi |Ih,i – Ih|/ΣhΣi Ih,i for the intensity (I) of observation i of reflection h. R factor = Σ‖Fobs| – |Fcalc‖/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree = R factor calculated using 5% of the reflection data chosen randomly and omitted from the start of refinement. RMSD, root mean square deviations from ideal geometry. Data for the highest-resolution shell are shown in parentheses.
FIG S1
Elution profile of NicA2(21–482) from Superdex 200 gel filtration chromatography, which suggests that NicA2 exists as a monomer in solution. The elution volume for the standard molecular weight marker is indicated above the chromatogram. Download FIG S1, TIF file, 0.5 MB.
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FIG S2
Structure of the FAD-binding domain of NicA2 with the bound FAD cofactor. The FAD-binding domain of NicA2 is shown as a cartoon representation and colored in yellow, with secondary-structure elements labeled. FAD is shown as a stick representation, with carbon, nitrogen, oxygen, and phosphorus atoms colored in cyan, blue, red, and orange, respectively. Download FIG S2, TIF file, 2.3 MB.
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FIG S3
Structure of the substrate-binding domain of NicA2 with the bound substrate nicotine. The S1 and S2 subdomains of the substrate-binding domain of NicA2 are colored in green and magenta, respectively. Secondary-structure elements of NicA2 are labeled. Nicotine is shown as a stick representation, with carbon and nitrogen atoms colored in yellow and blue, respectively. Download FIG S3, TIF file, 1.6 MB.
Copyright © 2020 Tang et al.
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FIG S4
The addition of either IPTG alone or nicotine alone did not affect the growth of P. putida S16 cells transformed with WT NicA2 or the 9AA mutant of NicA2. (A) The growth rate of P. putida S16 cells transformed with the empty pME6032 plasmid was not affected by the addition of either IPTG or nicotine alone. (B) The growth rate of P. putida S16 cells overexpressing WT NicA2 was not affected by the addition of either IPTG or nicotine alone. (C) The growth rate of P. putida S16 cells overexpressing the 9AA mutant of NicA2 was not affected by the addition of either IPTG or nicotine alone. Three duplicate samples were set for each group, and the bars represent standard deviations. Download FIG S4, TIF file, 0.7 MB.
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FIG S5
Overexpression of WT NicA2 also reduced the number of colonies of P. putida S16 on agar plates containing nicotine as the sole carbon source, while overexpression of the 9AA mutant of NicA2 had a more-severe effect and further inhibited the growth of colonies. On the other hand, coexpression of Pnao with the 9AA mutant of NicA2 rescued the growth of bacteria by alleviating the toxic effects of PN. Download FIG S5, TIF file, 3.0 MB.
Copyright © 2020 Tang et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
