Selenoxidation by Flavin-Containing Monooxygenases as a Novel Pathway for β-Elimination of Selenocysteine Se-Conjugates

Abstract

Previously, it was shown that beta-elimination of selenocysteine Se-conjugates by rat renal cytosol leading to pyruvate formation was not solely catalyzed by pyridoxal phosphate-dependent enzymes. It was hypothesized that selenoxidation of the selenocysteine Se-conjugates, followed by syn-elimination, may be an alternative mechanism for pyruvate formation. In this study, selenoxidation of selenocysteine Se-conjugates was studied using rat liver microsomes and recombinant human oxidative enzymes. For all six selenocysteine Se-conjugates that were tested, it was found that rat liver microsomal incubations led to the formation of pyruvate, whereas the corresponding selenoxides were not observed. Microsomal pyruvate formation from Se-benzyl-L-selenocysteine (SeBC) was NADPH-dependent, but only marginally inhibited by several P450 inhibitors. Inhibition by methimazole and by heat pretreatment and stimulation by n-octylamine indicated that flavin-containing monooxygenases are mainly responsible for pyruvate formation from the selenocysteine Se-conjugates in rat liver microsomes. In the case of S-benzyl-L-cysteine, the sulfur analogue of SeBC, pyruvate formation was not observed. For this substrate, a chemically stable sulfoxide could be observed, as previously described. By using recombinantly expressed human flavin-containing monooxygenases and P450 enzymes, it was delineated that SeBC is selenoxidized by human FMOs, but not by human P450s. The k(cat)/K(m) of selenoxidation was 3.8-fold higher for FMO-1 than for FMO-3. In conclusion, selenoxidation of selenocysteine Se-conjugates catalyzed by FMOs and subsequently syn-elimination has taken place as an alternative route for the formation of pyruvate from selenocysteine Se-conjugates. Although selenoxides are known to be easily reduced by thiol compounds, microsomal pyruvate formation from SeBC was only 75% inhibited in the presence of an excess of glutathione. This indicates that even in the presence of physiological concentrations of reducing thiol compounds, selenoxides of selenocysteine Se-conjugates may undergo syn-elimination to some extent. Whether selenoxides and/or selenenic acids that are formed are involved in the activity of chemopreventive selenocysteine Se-conjugates remains to be established.

Loss of selenium from selenoproteins: Conversion of selenocysteine to dehydroalanine in vitro

“…A similar mechanism was also proposed as a bio-activation pathway for pyruvate formation from the selenocysteine Se-conjugates in rat liver microsomes. Oxidation of selenocysteine Se-conjugates by flavin-containing monooxygenases resulted in spontaneous ␤-elimination of selenenic acid, leading to the formation of pyruvate and ammonia [17].…”

Liquid Chromatography/Tandem Mass Spectrometry Detection of Covalent Binding of Acetaminophen to Human Serum Albumin

“…The Acrodisc LC polyvinylidene difluoride filters (0.45 m, 25 mm) were obtained from Waters Corporation, and the Strata-X columns (33 m Polymeric Sorbent) were from Phenomenex. ␤-Naphthoflavone-induced rat liver microsomes (RLM) were prepared according to the standard protocol of our laboratory (Rooseboom et al, 2001). Control human blood was obtained from healthy volunteers, and blood samples from patients overdosed with APAP were provided to us by Dr. D. Touw (Apotheek Haagse Ziekenhuizen, The Hague, The Netherlands).…”

Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

“…A number of cysteine S-conjugates of xenobiotics are S-oxygenated by FMO (Table 3), but again the K m values are in the low mM range which may call into question the importance of such metabolism in vivo. The Se of selenocysteine conjugates is also oxygenated by FMO (Rooseboom et al, 2001). Interestingly, the sulfoxidation of farnesyl cysteine is catalyzed by FMO with a K m in the low μM range (Park et al, 1994).…”