The azoreductase of yeast cells: a new feature of an old enzyme

The azoreductase of yeast cells: a new feature of an old enzyme Patrícia A. Ramalho 1, Sandra Paiva 1, Artur Cavaco-Paulo 2, Margarida Casal 1, M. Teresa Ramalho 3, M. Helena Cardoso 1 1 Biology Department, 2 Textile Engineering Department, 3 Chemistry Department, University of Minho, Braga PORTUGAL

State of the art Reduction of azo dyes removes colour Yeasts are able to reduce azo dyes to amines Reduction of polar azo dyes occurs extracellularly Azo dye Ar-N=N-Ar? Colourless amines Ar-NH 2 + Ar -NH 2 0 h 5 h 10 h Oxidation products NAD(P)H NAD(P) + Yeast cell Carbon sources

From literature In yeasts an externally directed redox enzyme system - located in the plasma membran is the ferric reductase Ferric reductase activity is traditionally measured through the reduction of ferricyanide anion which is also impermeant to the plasma membrane This same activity is also seen in the reduction of ferric citrate or Fe/EDTA complexes Ferric reductase is involved in the iron uptake system of yeasts

Ferric reductase and iron uptake in yeasts Yeasts have three systems to solubilize ferric iron from the environment: - siderophores (Lesuisse et al., 2001); - excretion of reducing agents or organic acids (like anthranilate and 3- hydroxyanthranilate) (Bienfait et al., 1987; Georgatsou et al., 1994; Lesuisse et al. 1990); - reduction to Fe 2+ by a plasma membrane reductase (Lesuisse et al., 1990; Dancis et al., 1990). (In Shakoury-Elizeh et al., Mol. Biol. Cell, Mar 2004; 15: 1233 1243) ARN1-4: siderophore transporters FET4: low affinity ferrous iron transporter FET3+FTR1(permease): high affinity ferrous iron transporter ATX1: oxygen toxicity suppressor CCC2: iron transporter in the Golgi apparatus FIT: mannoproteins from the cell wall that retain iron

Methods Laboratorial Saccharomyces cerevisiae strain with the ability to reduce azo dyes Decolourization experiments along growth at 26ºC and 120 rpm Activity assays of ferric and azo reductases with cells grown for 6h in normal decolourization medium

Strategies to prove the new functionality of ferric reductase I. Measurement of both ferric and azo reductase activities along growth and decolourization: OD 640 nm 10 1 0.1 6 4 2 Dye Abs 461 nm / ph 0.01 0 Ferric and azo reductases have parallel activity curves with maxima in the late exponential growth phase Ferric reductase (μmol/(gdw*min)) 40 30 20 10 1.2 0.9 0.6 0.3 Azo reductase (μmol/(gdw*min)) 0 0 10 20 30 40 0 Time (h) D640 A461 ph Ferric reductase Azo reductase

Strategies to prove the new functionality A461 5 4 3 2 1 0 of ferric reductase II. Addition of an known inhibitor of ferric reductase (IRON) to the decolourisation medium: 0 20 40 60 80 Time (h) 0 Mm 1 mm 2.5 mm 5 mm The addition of iron to the medium inhibits ferric reductase at both transcriptional and posttranscriptional levels (Lesuisse et al. 1996) and delays decolorization

Strategies to prove the new functionality of ferric reductase III. Deletion of the genes FRE1 and FRE2 in the strain: effect on azo and ferric reductase activities 1.2 Relative activity 1 0.8 0.6 0.4 0.2 0 fre1δ fre1d fre2δ fre2d fre1δ fre1dfre2d fre2δ The deletion of FRE1 gene removes the decolorizing activity; FRE2 removal does not affect the decolorizing ability, in our conditions ferric reductase azo reductase

Strategies to prove the new functionality of ferric reductase III. Deletion of the genes FRE1 and FRE2 in the strain: effect on decolourisation activity OD 640 nm 10 1 0 10 20 30 40 50 60 Time (h) wt wt wt D OD OD fre1d fre1δdod fre2d fre2δd OD fre1dfre2d fre1δfre2δ D OD wt DyeAbs Abs fre1d fre1δdye DyeAbs Abs fre2d fre2δ Dye Dye Abs Abs fre1dfre2d fre1δfre2δdye DyeAbs Abs 5 4 3 2 1 0 Dye Abs 461 nm Reduction of decolourising capacity by deletion of FRE1 and FRE2 Fre1p is responsible for the major part of the azo reductase activity of intact yeast cells

Strategies to prove the new functionality IV. Use of inhibitors of ferric reductase CCCP (carbonyl cyanide m-chlorophenylhydrazone) protonofore (Schröder et al., 2003); DPI (diphenyleneiodonium chloride) flavoenzyme inhibitor (Wright and Kuhn 2002); Chloroquine antagonist of CoQ (Santos-Ocana et al., 1995); p-chloromercuribenzoate (pcmb) thiol reagent susceptible of reacting with intra-membrane protein groups; inhibits NADH:ferricyanide reductase (Löw et al., 1978).

Strategies to prove the new functionality IV. Use of inhibitors of ferric reductase Relative activity 3 2.5 2 1.5 1 0.5 0 0,01 mm CCCP 0,1 mm CCCP 0,010 mm DPI 0,2 mm CQ ferric reductase 0,01 mm pcmb 0,1 mm pcmb azo reductase 1mM pcmb The stimulation of the azoreductase activity points to the existence of two alternative paths and inhibition of the most favourable one. Both activities depend on a proton gradient Both depend on flavoenzymes Apparently CoQ x is more important in the azo reductase activity

Conclusions The ferric reductase is the main activity involved in extracellular azo dye reduction by yeasts (fre1p mainly). However there is an alternative redox system to ferric reductase in the plasma membrane still unknown and that is able reduce some extracellular compounds (azo dyes).

Proposed model Extracellular medium ArNH 2 Ar-N=N-Ar Fe(II) Ar-N=N-Ar ArNH 2 Fe(III) Fe(II) - (2) O A O 2 2 Fe(II) C Fe(III) Alternative Reductase Fre2p Fre1p [1] Electron (Q) Pool D DPI Plasma Membrane pcmb [2] NAD kinase [2] (UTR1) B Cytosol NADPH Dehydrogenase [1] NAD + NADP + NADPH NADP + The major fraction of azo reductase activity depends on Fre1p [1; this work] and on a NADPH dehydrogenase [1] A Activity of Fre1p depends on a cytosolic NAD kinase [2] B Azo dye reduction (an presumably ferric iron reduction) must occur at an alternative site [this work] C

Proposed model Extracellular medium ArNH 2 Ar-N=N-Ar Fe(II) Ar-N=N-Ar ArNH 2 Fe(III) Fe(II) - (2) O A O 2 2 Fe(II) C Fe(III) Alternative Reductase Fre2p Fre1p [1] Electron (Q) Pool D DPI Plasma Membrane pcmb [2] NAD kinase [2] (UTR1) B Cytosol NADPH Dehydrogenase [1] NADPH NADP + NAD+ NADP + pcmb stimulates azo reductase activity at higher concentrations [this work] These observations are consistent with the existence of membrane transporters capable of switching electrons between two external reduction sites (electron or Q pool) D [1] E Lesuisse, M Casteras-Simon and P Labbe 1996 JBC 271, 13578-13583 [2] S Kawai et al. 2001 FEMS Microbiol Lett 200, 181-184

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