Flavin Containing Monooxygenases FMOs EC 114138

Subcellular location: Membrane-bound to the cytoplasmic side of the ER (similar to P450 enzymes).

Organ distribution: Liver, kidney, intestine, lung, brain, skin, pancreas, and secretory tissue (see Table 2.10). Cofactor: NADPH.

Prosthetic group: Flavin adenine dinucleotide (FAD). Reaction: See Fig. 2.2.

Hydroperoxyflavin (FAD-OOH)

Hydroperoxyflavin (FAD-OOH)

Hydroxyflavin (FAD-OH)

Figure 2.2. FMO reaction mechanism (X=N, S, P and Se). FMO flavin-

containing monooxygenase.

Hydroxyflavin (FAD-OH)

Figure 2.2. FMO reaction mechanism (X=N, S, P and Se). FMO flavin-

containing monooxygenase.

Steps in the FMO reaction cycle:

1. At resting state, the enzyme is present as a hydroperoxyflavin (FAD-OOH; this form is stable). The FAD-OOH-activated intermediate can be considered a "cocked metabolism gun" because it is ready to react with a suitable substrate.

2. A nucleophilic substrate attacks the distal oxygen of FAD-OOH and results in formation of an oxygenated product and 4a-hydroxyflavin (FAD-OH).

3. The metabolite is released and FAD-OH losses water to form FAD. This step is thought to be rate-limiting.

4. FAD receives an electron from NADPH and is oxidized by O2 to form FAD-OOH. This step formally returns FMO to its resting state.

Forms (isoforms): There are five functional forms of FMOs (FMO1 to FMO5) in humans and six nonfunctional forms. FMO3 is the major form in the human liver. FMO1 is the major form in the many other animals, but is absent in human adult livers. FMO2 is lung selective. Apparently, FMO4 and FMO5 play minor roles in drug metabolism.

• FMO1 has a shallow substrate-binding channel and, therefore, a broad substrate specificity.

• FMO3 has a deep substrate-binding channel (8-10 A) and, therefore, a narrower specificity compared to FMO1.

Species and sex dependence in animal models. Pronounced differences in FMO expression exist in preclinical species (Janmohamed et al. 2004).

• Female mice have a very high expression of FMO3 and FMO5, which is considered to most closely resemble FMO expression in the adult human liver.

• Young female rats have higher FMO3 activity than do male rats (by five to tenfold).

• Adult rats have a relatively low FMO3 and FMO5 expression (unlike humans) and a constant FMO1 expression.

Table 2.10. FMO distribution in different species (Benedetti et al. 2006; Zhang and Cashman 2006)

Form

Mouse

Rat

Monkey

Human

FMO1

Kidney

Liver,

Kidney

Kidney » lung, small

kidney

intestine » liver

FMO2

Lung

Lung

Lung

Lung » kidney > liver,

small intestine

FMO3

Liver,

Kidney

Liver,

Liver » lung > kidney »

kidney

kidney

small intestine

Substrates: Imipramine (FMO1), cyclobenzaprine, chlorprom-azine, and nicotine (FMO3).

Inhibitors: Methimazole (also inhibits P450) and thiourea. No inhibitory antibodies are commercially available, but antibodies are available for Western Blot studies.

In humans, trimethylamine, a foul-smelling chemical derived from dietary sources such as choline and carnitine, is metabolized by FMO3 to an odorless N-oxide metabolite. FMO3 deficiency leads to trimethylaminuria ("fish-like odor syndrome").

FMO contribution to metabolism is usually underestimated because:

1. P450 enzymes usually generate the same metabolites.

2. Oxides (FMO or P450 metabolites) can be reduced to the parent molecule. This process is called "retro-reduction" (Cashman 2008).

3. FMOs are thermally unstable in the absence of NADPH and, therefore, can be inactivated in a preincubation step if done at 37°C (Fig. 2.3).

Cope-type elimination

O Cope-type oh

Oxime formation

O enzymatically or non-enzymatically

Carbon oxidation

Figure 2.3. Examples of unusual FMO reactions: (a) some N-oxides are not stable, especially 3o N-oxide, and lead to Cope-type elimination, (b) oxime formation, and (c) carbon center oxidation formation of 4-hydroxylaniline from 4-fluoro-N-methylaniline (Driscoll et al. 2010). FMO flavin-containing monooxygenase.

Useful chemicals:

• Diethyleneamine tetra-acetic acid (DETAPAC) is used to minimize autooxidation in microsomal incubations.

• meta-Chloroperoxybenzoic acid (mCPBA) is useful for synthesizing oxides.

• TiCl3 is used to reduce FMO S- and N-oxide metabolites.

FMOs vs. P450 enzymes

Both enzymes reside in liver microsomes (LM) and require O2

and NADPH for their activity but:

1. Optimum pH: >9 for FMOs and 7.4 for P450 enzymes.

2. Most FMOs are thermally unstable when preincubated in the absence of NADPH. This is true for FMO1, FMO3, FMO4, and FMO5, but not FMO2.

3. Chemical inhibitors: Methimazole inhibits FMOs (except for FMO5) and P450 enzymes. ABT selectively inactivates P450 enzymes in a time-dependent manner, but does not inhibit FMOs.

4. Inhibitory antibodies are not available for FMOs, but are available for P450 isoforms. Antibodies to P450 can be used to distinguish between FMO-dependent and P450-dependent processes.

5. Detergents (such as Triton X-100) have little effect on FMO activity, but they inhibit P450 enzymes.

6. Recombinant enzymes can be used to distinguish between FMO- and P450-catalyzed reactions.

7. FMOs are rarely induced, but P450s are inducible.

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