Velocity rates for coumarin 7-hydroxylation in incubations of coumarin (10 µM) without (open bars) or with (hatched bars) pre-incubation with 8-methoxypsoralen at 1.0 µM in CYP2A6 proteins.

<p>Data are shown as mean ± SD from triplicate determinations. Degree of statistical differences between incubations without and with pre-incubation as determined by two-tailed Student's <i>t</i> test: the mean difference is significant at 0.05 level (* p<0.05) and highly significant at 0.01 level (** p<0.01).</p

Geometric changes in CYP2A6 wild type and mutant proteins.

<p><b>a)</b> Changes in the geometry of the protein tertiary structures. Wild type, red; CYP2A6*15, green; CYP2A6*16, blue; CYP2A6*21, black; CYP2A6*22, magenta; <b>b)</b> Superimposition of the wild type and mutant structures. Yellow circle indicates the binding site.</p

Binding of 8-methoxypsoralen in CYP2A6 wild type active site.

<p>Side chains of amino acid residues within 5 Å of the ligand are rendered as stick figures. Red, oxygen atoms; blue, nitrogen atoms; gray, methoxsalen-complex carbon atoms; and magenta, heme group.</p

Conformational changes of the CYP2A6 active site cavities.

<p><b>a)</b> Space-filling representation of amino acid residues forming the CYP2A6 active sites. a1: wild type; a2: CYP2A6*15; a3: CYP2A6*16; a4: CYP2A6*21; and a5: CYP2A6*22. <b>b)</b> The conformations of active site cavities depicted as stick models. b1: wild type; b2: CYP2A6*15; b3: CYP2A6*16; b4: CYP2A6*21; and b5: CYP2A6*22. The heme moiety is presented at the floor of active sites as magenta sticks.</p

Representative IC₅₀ plots demonstrating the inhibitory effect of 8-methoxypsoralen at concentration of 0–5 µM on CYP2A6*1 (wild type) (•), CYP2A6*15 (▴), CYP2A6*16 (▪), CYP2A6*21 (♦) and CYP2A6*22 .

<p>Points are experimentally determined values while solid lines are the computer-generated curves of best fit (by SigmaPlot® programme). Data are presented as mean values from triplicate measurements.</p

Binding of 8-methoxypsoralen in CYP2A6 mutant active sites.

<p>Side chains of amino acid residues within 5 Å of the ligand are rendered as stick figures. Red, oxygen atoms; blue, nitrogen atoms; gray, methoxsalen-complex carbon atoms; and magenta, heme group. a: CYP2A6*15; b: CYP2A6*16; c: CYP2A6*21; and d: CYP2A6*22.</p

The ribbon diagram showing the overall structure of the wild-type CYP2A6 with locations of mutated residues highlighted.

<p>Secondary structures α-helices and β-sheets are also shown and labelled. The heme moiety is indicated in sticks mode (magenta color).</p

The kinetic and inhibition profile of 8-methoxypsoralen on coumarin 7-hydroxylase activity of the wild type CYP2A6, CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22.

<p>All values are shown as the means ± SD of three independent determinations. The K_m values were determined in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086230#pone.0086230-Tiong1" target="_blank">[9]</a> and listed here for comparison purpose. The IC₅₀ values were determined in the present studies. The last column represents the fold of change in IC₅₀ relative to the wild type. Degree of statistical differences between the wild-type and the mutants as determined by one-way analysis of variance: the mean difference is significant at 0.05 level (* p<0.05) and highly significant at 0.01 level (** p<0.01).</p