Proposed pathways for nicotine degradation in Pseudomonas, Arthrobacter, and eukaryotes. A, in Arthrobacter nicotinovorans. B, in Pseudomonas. The reaction in the dashed frame is catalyzed by the HSP hydroxylases, HspA and HspB. C, in Aspergillus oryzae.

Purification of HspB. A, SDS-PAGE. Lane M, marker proteins; lane 1, P. putida S16 cell extract; lane 2, supernatant from (NH₄)₂SO₄ precipitation; lane 3, source 30Q pool; lane 4, source 15ISO pool; lane 5, Mono Q fraction; lane 6, Sephacryl S-200 HR fraction. B, native PAGE of purified HspB. Lane M, native protein marker; lane 1, purified HspB. C, three-dimensional plot of HPLC analysis for FAD from the boiled HspB solution.

Characterization of HspB. A, pH dependence of HspB stability. The enzyme was incubated overnight in buffers of various pH at 25 °C. B, thermostability of HspB. The enzyme was incubated in Tris-HCl buffer (50 mm, pH 8.0) at varying temperatures for 30 min. C, effects of metal salts on HspB stability. CK, without metal salt; Na, Na⁺; Ni, Ni²⁺; Co, Co²⁺; Ca, Ca²⁺; Cu, Cu²⁺; Mn, Mn²⁺; Zn, Zn²⁺; Mg, Mg²⁺; K, K⁺; Mo, MoO₄²⁻; W, WO₄²⁻. D, oxygen consumption of the HspB reaction. ▲, ●, and ■ indicate the oxygen declines from different initial concentrations. For the above four experiments, enzyme activity was determined according to the standard assay at 25 °C and pH 8.0.

Mass spectra of DHP as determined by LC-MS analysis. The mass spectra of derived DHP are obtained from reactions without ¹⁸O₂ (A) and with ¹⁸O₂ (B). The positions of the peaks for [¹⁸O] DHP are indicated by arrows.

Mass spectrum of succinic semialdehyde as determined by GC-MS analysis. The GC-MS determination with derivatization of silanization by special peak is as follows. A molecular ion peak is seen at m/z 174, and its fragments are at m/z 29 ([CHO]⁺), 45 ([COOH]⁺), 89 ([Si(CH₃)₃O]⁺), and 73 ([(CH₂)₂COOH]⁺).

Cell growth and resting cell reactions of strain S16 and the gene deletion mutants. A, utilization of nicotine for growth by S16 hspA::pk18mob (deletion of hspA), S16 (as control), and S16 hspB::pk18mob (deletion of hspB). ■, cell growth of S16 hspA::pk18mob (deletion of hspA); ●, cell growth of S16 as control; ▲, cell growth of S16 hspB::pk18mob (deletion of hspB); □, nicotine concentration in the medium for S16 hspA::pk18mob growth; ○, nicotine concentration in the medium for S16 growth as control; △, nicotine concentration in the medium for S16 hspB::pk18mob growth. B, resting cell reactions of S16 hspA::pk18mob (deletion of hspA), S16 (as control), and S16 hspB::pk18mob (deletion of hspB) in nicotine degradation. ■, nicotine concentration in the medium for S16 hspA::pk18mob degradation; ●, nicotine concentration in the medium for S16 degradation as control; ▲, nicotine concentration in the medium for S16 hspB::pk18mob degradation. C, resting cell reactions of S16 hspA::pk18mob (deletion of hspA), S16 (as control), and S16 hspB::pk18mob (deletion of hspB) in HSP degradation. ■, HSP concentration in the medium for S16 hspA::pk18mob degradation; ●, HSP concentration in the medium for S16 degradation as control; ▲, HSP concentration in the medium for S16 hspB::pk18mob degradation. The values are the means of three replicates, and the error bars indicate the standard deviations.