Single mutations change CYP2F3 from a dehydrogenase of 3-methylindole to an oxygenase

Abstract

Pulmonary cytochrome P450 2F3 (CYP2F3) catalyzes the dehydrogenation of the pneumotoxin 3-methylindole (3MI) to an electrophilic intermediate, 3-methyleneindolenine, which is responsible for the toxicity of the parent compound. Members of the CYP2F subfamily are the only enzymes known to exclusively dehydrogenate 3MI, without detectable formation of oxygenation products. Thus, CYP2F3 is an attractive model to study dehydrogenation mechanisms. The purpose of this study was to identify specific residues that could facilitate 3MI dehydrogenation. Both single and double mutations were constructed to study the molecular mechanisms that direct dehydrogenation. Double mutations in substrate recognition sites (SRS) 1 produced an inactive enzyme, while double mutants in SRS 4 did not alter 3MI metabolism. However, double mutations in SRS 5 and SRS 6 successfully introduced oxygenase activity to CYP2F3. Single mutations in SRS 5, SRS 6 and near SRS 2 also introduced 3MI oxygenase activity. Mutants S474H and D361T oxygenated 3MI but also increased dehydrogenation rates, while G214L, E215Q and S475I catalyzed 3MI oxygenation exclusively. A homology model of CYP2F3 was precisely consistent with specific dehydrogenation of 3MI via initial hydrogen atom abstraction from the methyl group. In addition, intramolecular kinetic deuterium isotope studies demonstrated an isotope effect ( K H/ K D) of 6.8. This relatively high intramolecular deuterium isotope effect confirmed the initial hydrogen abstraction step; a mutant (D361T) that retained the dehydrogenation reaction exhibited the same deuterium isotope effect. The results showed that a single alteration, such as a serine to isoleucine change at residue 475, dramatically switched catalytic preference from dehydrogenation to oxygenation.

Figure 1

The aligned protein sequences of CYP2F3 and CYP2E1 and mutations. The numbers above the sequences correspond to the CYP2F3 sequence, and the numbers below the sequences correspond to the CYP2E1 sequence. The single and double mutations that were successfully mutated to produce active enzymes are shown in large bold type. The double mutations (both residues mutated simultaneously) are boxed. The six substrate recognition sites are indicated with bold type. All mutants were changed from the 2F3 sequence to the corresponding 2E1 residues.

Figure 2

Thiol trapping of oxygenation- and dehydrogenation-dependent reactive intermediates of 3-methylindole. Pathways 1a, 1b, and 1c illustrate formation of the dehydrogenated reactive intermediate, 3-methyleneindolenine, and trapping of the electrophile with NAC. X = H corresponds to 3MI, X = D corresponds to 3MI-d3, and X = HD₂ corresponds to 3MI-d2. [2-²H]-3-Methylindole is the deuterium-labeled substrate for pathways 2a and 3a. Pathways 2a, 2b, 2c, 2d, and 2e illustrate formation of the oxygenated reactive intermediate, 3-hydroxy-3-methylindolenine, and trapping of the electrophile with NAC or thioglycolic acid. 3-Methyloxindole is formed from ring opening and hydride shift of the epoxide (6). Pathways 3a, 3b, and 3c illustrate formation of the dehydrogenated intermediate, 3-methyleneindolenine, and trapping with thioglycolic acid. Pathway 1a→1b - Formation of 3-methyleneindolenine from 3MI, followed by nucleophilic attack by N-acetyl-L-cysteine at the 2 position to form the C-2 adduct. Pathway 1a→1c - Formation of 3-methyleneindolenine from 3MI, followed by nucleophilic attack by N-acetyl-L-cysteine at the 3 position to form 3MINAC. Pathway 2a→2b→2c — Formation of the C-2 adduct by ring opening of the 2,3-epoxide to form 3-hydroxy-3-methylindolenine, followed by nucleophilic attack at the 2-position by N-acetyl-L-cysteine and subsequent dehydration with the loss of the C-2 deuterium. Pathway 2a→2b→2d - Thioglycolic acid trapping of the oxygenation-dependent reactive intermediate 3-hydroxy-3-methylindolenine and cyclic esterification of the conjugate to form a thiolactone adduct. The cyclic thiolactone adduct retained the C-2 deuterium. Pathway 2a→2b→2e — Trapping of the oxygenated metabolite, 3-hydroxy-3-methylindolenine with thioglycolic acid to form a 2-position adduct that dehydrates to aromatize with a loss of HOD. Pathway 3a→3b - Thioglycolic acid trapping of the dehydrogenated intermediate and formation of the 2-position thioglycolic adduct. The adduct loses the C-2 deuterium during tautomerization to aromatize. Pathway 3a→3c- Thioglycolic acid trapping of the dehydrogenated intermediate and formation of the 3-position thioglycolic adduct. The adduct retains the C-2 deuterium.

Figure 3

Representative total ion chromatogram for incubations of CYP2F3 with [2-²H]-3-methylindole and thioglycolic acid. Closely eluting peaks with m/z ratios of 223 (RT: 10.3 and 10.6 minutes) were the diastereomers of the cyclic thiolactone adduct of 3-hydroxy-3-methylindolenine. The peak with an m/z of 222 (RT: 15 minutes) was the C-2 adduct, formed either by trapping of the oxygenated metabolite, 3-hydroxy-3-methylindolenine, or trapping of 3-methyleneindolenine. The thioglycolic acid adduct with an m/z of 223 (RT: 14.7) was most likely an adduct at the exocyclic carbon of the methylene imine. BP = molecular ion

Figure 4

Kinetics of formation of 3MI metabolites by 2F3 and the mutant enzymes. Data points represent the mean of 3 separate experiments. The X-axis is 3MI concentration in micromolar. The Y-axis is nmol product formed/min/nmol P450

Figure 5

Homology model of P450 2F3 (A) and docking of substrate, 3MI, in the active site (B). A, all secondary structural α-helices and β-strands are shown in green and are labeled. Also shown are SRS 1 in magenta, SRS 2 in orange, SRS 3 in marine, SRS 4 in yellow, SRS 5 in red, and SRS 6 in blue. The heme is represented with sticks and is colored cyan. B, all secondary structures are portrayed with a transparent cartoon in the background, with the heme, 3MI, and important active site residues shown as colored sticks (the heme is pink, 3MI is green, SRS 5 mutated residues A360 and D361 are yellow, SRS 6 mutated residues S474 and S475 are orange, mutated residues G214 and E215 are red, residues I362 and I363 are cyan, Y101 is pink, and F206 is blue). The figures were produced with PyMOL.

Figure 5

Homology model of P450 2F3 (A) and docking of substrate, 3MI, in the active site (B). A, all secondary structural α-helices and β-strands are shown in green and are labeled. Also shown are SRS 1 in magenta, SRS 2 in orange, SRS 3 in marine, SRS 4 in yellow, SRS 5 in red, and SRS 6 in blue. The heme is represented with sticks and is colored cyan. B, all secondary structures are portrayed with a transparent cartoon in the background, with the heme, 3MI, and important active site residues shown as colored sticks (the heme is pink, 3MI is green, SRS 5 mutated residues A360 and D361 are yellow, SRS 6 mutated residues S474 and S475 are orange, mutated residues G214 and E215 are red, residues I362 and I363 are cyan, Y101 is pink, and F206 is blue). The figures were produced with PyMOL.