Yeast flavin-containing monooxygenase generates oxidizing equivalents that control protein folding in the endoplasmic reticulum

Suh, Jung Keun; Poulsen, Lawrence L.; Ziegler, Daniel M.; Robertus, Jon D.

doi:10.1073/pnas.96.6.2687

Cited by 78 publications

(69 citation statements)

References 27 publications

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“…In eukaryotes many proteins are synthesized and undergo posttranslational modification in the ER. These processes require an oxidized environment, which is re- flected in a relatively low GSH:GSSG ratio (approximately 2:1 compared with approximately 50:1 in the cytosol) and the presence of enzymes that produce oxidizing compounds (38)(39)(40). Paradoxically, in the ER and associated vesicular structures, millimolar levels of ascorbate have been reported (41,42).…”

Section: Discussionmentioning

confidence: 99%

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Wilkinson

Obado

Maurício

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

116

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In most aerobic organisms hemoperoxidases play a major role in H 2O2-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H 2O2. To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.

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Section: Discussionmentioning

confidence: 99%

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Wilkinson

Obado

Maurício

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

116

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“…Cysteamine is one of a few endogenous substrates for FMO (see Section 2.4) and is oxidized to the disulfide cystamine. Interestingly, the yeast FMO has been shown to carry out such a cellular function, i.e., it functions in disulfide bond formation in protein synthesis and regulates thiol/disulfide ratios in the cell (Suh et al, 1999;Suh & Robertus, 2002). The yeast FMO gene 5′-promoter region contains an unfolded protein response element and transcription is regulated by this pathway .…”

Section: Introductionmentioning

confidence: 99%

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

Krueger

Williams

2005

Pharmacology & Therapeutics

515

444

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Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a "softnucleophile", usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics.In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences.The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration.

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“…The FGly-forming enzyme is a component of the ER lumen where an oxidizing environment is maintained by Ϸ1 mM glutathione (reduced (GSH) plus oxidized (GSSG) glutathione) with a GSH/GSSG ratio of 1:1 to 3:1 (25). To test for a possible influence of glutathione, we investigated FGly modification using reticuloplasm from which all low molecular weight components were removed by gel filtration (see "Experimental Procedures").…”

Section: Fig 2 Fgly Modification By a Microsomal Detergent Extractmentioning

confidence: 99%

Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

Fey¹,

Balleininger²,

Borissenko³

et al. 2001

Journal of Biological Chemistry

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C ␣ -formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is Ϸ10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca 2؉ . Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.

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Yeast flavin-containing monooxygenase generates oxidizing equivalents that control protein folding in the endoplasmic reticulum

Cited by 78 publications

References 27 publications

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

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

Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

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