In Vivo Metabolism of l-Methionine in Mice: Evidence for Stereoselective Formation of Methionine-d-Sulfoxide and Quantitation of Other Major Metabolites

Abstract

Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-and O 2 -dependent oxidation of heteroatoms (nitrogen, sulfur, phosphorus) present in the chemical structure of a variety of drugs and xenobiotics. Five functional forms (FMO1-5) have been characterized to date (Ziegler, 2002). In humans, FMO1 is the primary isoform expressed in neonate liver, but a switch occurs shortly after birth to FMO3, the primary isoform expressed in adult human liver (Koukouritaki et al., 2002). In mice, females, but not males, also express FMO3 in the liver (Falls et al., 1995;Ripp et al., 1999b). This sex-specific expression of FMO3 in mice makes them an attractive model for studying the role of FMO3 in human drug metabolism and disease.Our laboratory first identified Met as a substrate for cDNA-expressed rabbit FMO1-3 with K m values of 48.0, 29.9, and 6.5 mM, respectively (Duescher et al., 1994). The V/K values (0.9, 1.7, and 6.1 for FMO1-3, respectively) also suggested that FMO3 was the most efficient Met S-oxidizer of these three FMO isoforms. FMO3 Soxidation of Met was highly stereoselective, forming 8.4 times more methionine-d-sulfoxide (Met-d-O) than methionine-l-sulfoxide (Metl-O), whereas the d:l diastereomeric ratios for FMO1 and FMO2 were 1.5:1 and 0.7:1, respectively; FMO5 S-oxidation of Met was not detected. Recombinant human FMO3 exhibited a K m value of 3.7 mM with a V/K value of 4.6 and resulted in stereoselective formation of (Ripp et al., 1999b). Recombinant human FMO4 S-oxidation of Met exhibited a K m value greater than 10 mM with only 30% of the total sulfoxide formed being Met-d-O (Ripp et al., 1999a). The V/K value for FMO4 was not determined. These data indicated that FMO3 S-oxidation of Met proceeds with the highest affinity and greatest diastereomeric selectivity among FMO1-5.Stereoselective formation of Met-d-O was also detected in rabbit and rat liver microsomes incubated with Met, and exhibited K m and V/K values similar to those of cDNA-expressed FMO3 (Duescher et al., 1994;Krause et al., 1996). The latter results provided evidence for FMO3 being the primary isoform involved in Met S-oxidation in rabbit and rat liver. Additional experiments also provided evidence that FMOs, but not cytochrome P450s, peroxidases, or reactive oxygen species, mediated the Met S-oxidase activity (Krause et al., 1996).

l-Methionine Toxicity in Freshly Isolated Mouse Hepatocytes Is Gender-Dependent and Mediated in Part by Transamination

“…Gender differences in Met metabolism such as increased Met TM and/or decreased Met TA in females could also contribute to the observed toxicological differences. In support of this hypothesis, Met-dosed female mice had higher levels of SAM present in the liver after 15 min compared with Met-dosed males (Dever and Elfarra, 2006). Further investigations are required to examine potential gender differences in 3-MTP metabolism and rates of Met TM and Met TA in mouse hepatocytes.…”

The biological significance of methionine sulfoxide stereochemistry

“…From what we currently know, there is no evidence for preferential formation of one diastereomer of Met sulfoxide over the other in proteins and free amino acid forms, with the exception of enzymatic oxidation by flavin-containing monooxygenase 3 (26, 27). Prior to the discovery of Met- R -SO specific reductase, researchers focused on characterizing stereospecific oxidation by ROS because it was known that oxidized Met residues could be completely reduced in vivo , whereas only a Met- S -SO specific reductase was known at the time.…”

Regulated methionine oxidation by monooxygenases

“…the nonproductive decay of the hydroperoxideintermediate 59,65–68 (Figure 2, step e). While mammalian FMOs have been extensively characterized as detoxifying enzymes, it has been shown that FMO1, FMO2, and FMO3 possess catalytic activity toward methionine, generating MetO 56,69–72 . Interestingly, the MetO formed in liver, kidney, and lung microsomal fraction shows enrichment for the R isomer, pointing to a catalytic mechanism rather than a direct reaction with H 2 O 2 .…”