Despite the crucial roles of flavin nucleotides in metabolism, we know little about the enzymes responsible for turnover of FMN and FAD and their subcellular localization. Growing cells depend on a continuous supply of newly synthesized FMN and FAD which are required as cofactors for a large number of flavoenzymes. The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial role in both bioenergetics and cellular regulation [1]. The homeostasis of riboflavin and its prosthetic groups may be altered by some factors, such as defective FMN and/or FAD synthesis, increased catabolism, by different susceptibility of holo- and apo-flavoproteins to proteolytic digestion and altered mitochondrial metabolism and transport. Previous studies have already reported that isolated Saccharomyces cerevisiae mitochondria (SCM), can take up externally added riboflavin and synthesise FAD. This has been assumed to occur via riboflavin kinase and FAD synthetase which catalyse FMN synthesis from riboflavin and ATP and FAD from FMN and ATP respectively [2]. Moreover, SCM can export newly synthesised flavin derivatives to the extramitochondrial phase [3], presumably through the Flx1p transporter [4]. In this report, the experimental work was articulated in the following phases: 1. In a series of preliminary experiments SCM were checked with respect to their functional features (intactness of mitochondrial outer membrane, intactness of mitochondrial inner membrane, Respiratory Control, ΔΨ generation) 2. Then, the amounts of flavins in aliquots of neutralized perchloric extracts of both spheroplasts and mitochondria (pellets and supernatants) were measured by HPLC 3. SCM capability to metabolise externally added and endogenous FAD and FMN was investigated. Experiments were undertaken both spectroscopically and via HPLC. FAD deadenylation and FMN dephosphorylation were studied with respect to following features: dependence on substrate concentration, pH profile and inhibitor sensitivity. The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (diphosphatase) (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD + H2O→FMN + AMP and FMN + H2O →riboflavin + Pi conversion, respectively, is here shown. In the light of cytosolic riboflavin, FMN and FAD concentrations, as calculated by measuring both spheroplast and mitochondrial content via HPLC, probably mitochondria play a major role in regulating the flavin pool in yeast cell.

Saccharomyces cerevisiae as a model system for studying mitochondria natural flavin catabolism

PALLOTTA, Maria Luigia
2008

Abstract

Despite the crucial roles of flavin nucleotides in metabolism, we know little about the enzymes responsible for turnover of FMN and FAD and their subcellular localization. Growing cells depend on a continuous supply of newly synthesized FMN and FAD which are required as cofactors for a large number of flavoenzymes. The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial role in both bioenergetics and cellular regulation [1]. The homeostasis of riboflavin and its prosthetic groups may be altered by some factors, such as defective FMN and/or FAD synthesis, increased catabolism, by different susceptibility of holo- and apo-flavoproteins to proteolytic digestion and altered mitochondrial metabolism and transport. Previous studies have already reported that isolated Saccharomyces cerevisiae mitochondria (SCM), can take up externally added riboflavin and synthesise FAD. This has been assumed to occur via riboflavin kinase and FAD synthetase which catalyse FMN synthesis from riboflavin and ATP and FAD from FMN and ATP respectively [2]. Moreover, SCM can export newly synthesised flavin derivatives to the extramitochondrial phase [3], presumably through the Flx1p transporter [4]. In this report, the experimental work was articulated in the following phases: 1. In a series of preliminary experiments SCM were checked with respect to their functional features (intactness of mitochondrial outer membrane, intactness of mitochondrial inner membrane, Respiratory Control, ΔΨ generation) 2. Then, the amounts of flavins in aliquots of neutralized perchloric extracts of both spheroplasts and mitochondria (pellets and supernatants) were measured by HPLC 3. SCM capability to metabolise externally added and endogenous FAD and FMN was investigated. Experiments were undertaken both spectroscopically and via HPLC. FAD deadenylation and FMN dephosphorylation were studied with respect to following features: dependence on substrate concentration, pH profile and inhibitor sensitivity. The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (diphosphatase) (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD + H2O→FMN + AMP and FMN + H2O →riboflavin + Pi conversion, respectively, is here shown. In the light of cytosolic riboflavin, FMN and FAD concentrations, as calculated by measuring both spheroplast and mitochondrial content via HPLC, probably mitochondria play a major role in regulating the flavin pool in yeast cell.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11695/14264
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