FEMS kticrobiology Letters 7 (1980)55-59 © Cope, right Federation of European Microbiological Societies Published by Elsevier/North-Holland Biomedical Press

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DIMETHYL SULPHOXIDE REDUCTASE OF SACCHAROMYCES SPP.

C.W. BAMFORTH

Brewb~g Research Foundation, Nutfield, Surrey, Rtll 4HY, U.K.

Received t6 October 1.979 Accepted 26 October 1979

1. Introduction

Dimethyl sulphide (DMS) is an important flavour constituent of beers [1 ], especially lagers [2]. It can be formed through the reduction by yeast of dimethyl sulphoxide (DMSO)which is preseht to a small extent in barley and green malt but which is produced in increased quantities during malt kilning [3]. Washed cell suspensions of Saecttaromyces spp. convert DMSO to DMS and crude extracts contain a consti- tutive, NADPH-specific, DMSO reductase [3,4]. Fur- therrnore brewery spoilage bacteria can sometimes contribute to the DMS content of beers [5 ] by reducing DMSO [6].

DM$, arising chiefly from algal metabolism, has been implicated as the natural compound responsible for the transfer of sulphur from the seas, through air, to land systems [7]. The atmospheric photochemical oxidation of DMS to DMSO is an intermediate stage in this process, Also DMSO is artifically introduced to the biosphere as a solvent to increase membrane permeability to nutrients and pesticides. Thus the significance of microbial DMSO reduction is not restricted to brewing. The conversion of DMSO to DMS by a range of prokaryotic and eukaryotic micro- organisms has been shown [g~lO] and a DMSO reductase was identified in Escherichia colt which could use NADH or NADPH as electron donor [9].

Other naturally-occurring sulphoxides include me- thiornine sulphoxide (MetSO) [11 ] and biotin sul- phoxide (BioSO) [121, probably arisbag from their corresponding sulphides bY peroxidation [13]. En- zyme systems have been described catalyzing the reductior~ of bletSO in yeast [14] and g. coli [15] and Bio$O in E. coli [16].

The present paper present evidence which suggests that DMSO reductase in yeast is similar to MetSO reductase and confirms that, in contrast, bacteria possess a DMSO-reducing enzyme wich has highest activity with NADH.

2. Mate~h and Methods

2.1. Chemicals

Special chemicals were obtained from Sigma (London) Chemical Co., except for DMSO which was obtained from Koch.Light Laboratories Ltd. Biotin sulphoxides were prepared as described by Melville [12],

2.2. Organisms and cultivation

Yeasts from the National Collection of Yeast Cul- tures (NCYC) were grown either on glucose-salts medium as described previously [3] of on Difeo YM broth. In the latter case cultures were grown in 400 nd batches in Roux bottles shaken at 250C. Cells were harvested at middle to late exponential phase, washed once in deionised water and stored at - I $°C, For the preparation of enzyme fraction B Saccharo- myees cere~isiae (NCYC 240) grown in a brewery fermentation was used. In this case the extract was produced by freezing the yeast at -iS°C, subse- quently thawing and centrifuging (1000 ×g, 20 mia, room temperature). The resultant supernatant was used as enzyme source, lit was concentrated by taking to 90% saturation with (Nl'hhSOa and fedissoleing the precipitate in one-tenth of the origfnal volume

56

of extract. All bacteria were isolated as brewery con- taminants. E. coli and l;nterobacter cloacae were grown without shaktttg on Oifco YM broth at 37°C. Flavobacterium proteus was grown on this medium with shaking at 25"C. Cells were harvested after 24 h, washed once in deionised water and stored at -150C.

2.3. Preparation of cell.free extracts

Yeast or bacteria were suspended in a volume of 299 mM potassium phosphate, pH 7.0 (containing 2 raM 2-mercaptoethanol when disintegrating bacteria [9]) equal to the volume of cell paste, Tl~e cooled suspension was mixed witit ballotini beads (0.44- 0.53 ram) (5 g per ml suspension) and shaken at 2000 rev.]min for 1 rain in a cooled homogenizer (B. Braun, Melsungen). Ballotini beads were removed by vacuum filtration and the filtrate centrifuged at 25 000 ×g for I0 rain at 40C. ~cterial extracts were dialysed against 20 vol. of 50 mM potassium phos. phate, pH 7.0 at 4°C for 4 h before use.

2.4. Enzyme assays and protein estimation

Yeast DMSO reduetase was assayed using 25 ml McCartney bottles containing DMSO, 2 Vanol; NADPH, 0.5 mac1; enzyme and 200 mM potassium phosphate, pit 7.0 to 1.4 ml. Bottles were capped w/th Suba Seal stoppers and submerged in a water bath at 30*C. Reaction was initiated by addition of enzyme and DMS produced measured after 30 rain by g.l.c, of samples (2 ml) of headspace [3]. Control assays omitted DMSO, NADPH or enzyme. DMS was fully partitioned into the vapour phase within I0 rain.

Bacterial DMSO reductase was assayed similarly except that NAD(P)H was used to initiate the reac- tion. Prior to incubation at 30°C bottles were flushed for 3 rain with O2-free N2.

MetSO reductase (EC 1.6.99.-) was detem~ined at 30°C by speetrophotometri¢ measurement at 340 nm of MetSO.dependent oxidation of NADPH. Cuvettes (1 ¢m path length) contained potassium phosphate, pH 7.0, 20/amol; bovine serum albumin (BSA), 2 rag; NADPH, 0.25/areal; enzyme and water to a final volume of 1.5 ml. After measuring the rate of the endogenous NADPH oxidation, the reaction was started by adding MetSO, 5/,tmol.

Thioredoxin/thioredoxin reductase (EC 1.6.4.5)

were determined together at 30*C by following the increase inA4t~ due to the reduction of 5,5'.ditMobis- (2.nitrobertzoio acid) (DTNB). 1 cm path length cuvettes contained NADPH, 0.25/Jmol; EDTA, 20 Vinci; DTNB, 0.4 Vinci; enzyme and lO0 mM potassium #osphate, pH 7.0 to 1.5 ml. R~action was started by the addition of enzyme,

Protein was estimated from UV absorbance at 230 and 260 nm [17].

3. Results

3.1. DMSO reduction by crude extracts of~¢charo. myces sp.

Crude extracts of$. eerevisiae (NCYC 240) catalyze the NADPH.dependent reduction of DMSO [3,4]. NADPH cannot be replaced by NADH or ascorbate plus 3,6-dichloro phenol-indo phertol [4]. Substituting an N2-gas phase for air had no effect. Replacement of NADPH with 2-mercaptoetbanol (10/anol) gave 53% of the activity found with NADPH after correction for DMSO reduction by mercaptoethanol in the absence of enzyme. This suggests that a on.factor is present itt the extract which is capable of reduction by mercaptoethanol, of. reduction of thioredoxin by dithiothreitol [18] or dithioerythritol [19]. Although NADPH-linked activity was proportional to assay time it was not proportional to protein concentration (Fig. 1). The observed exponential increase in activity suggests a multi-protein system is involved.

After c~trifugation at 150000 Xgfor I h all DMSO reductase activity was located in the super- natant fraction, indicating that it is a soluble enzyme. Recombination with the membrane fraction did not simulate DMS formation.

DMSO reductase in crude extrv~ ~ ~'2.I mg protein rnl -t) was unstable, losing 47% ali~ 72% of its activity on overnight storage at -15OC and 4'~C respectivelY. The enzymewas less stable in the presen~ of 1 mg ml-t BSA, 2 mM mercaptoethanol or 250 ~ NADPH but increasir/gthe potassium phosphate OH 7.0) con- centration from 50 to 150 mM caused retention of I l 1: and 66% activity on keeping overnight at -15°C and 4°C, respectively.

L.MetMonine.dl.suiphoxide is a competitive inhi- bitor of DMSO reductase in crudeextmcts [4].

'°°[ aok,

oMs c~

6C

4C

2C • a

0=. .. I 0 1 2 3 4 5

pro~n ( ~ Fig. I . Effect o f protein concentration in crude extracts o f Saccharomyces cerel,isiae (NCYC 240) on DMSO tedurtas¢ activity, Assay was performca as described in Methods,

Neither t~-biotind-sulphoxide (20 raM) riot V-biotin4- sulphoxide (6.7 raM) was inhibitory.

NADPHdinked/DMSO reductase was preseni at similar specific activities ~ all strains of brewers' yeast examined. Thus the specific activities in S. cere- visiae NCYC 240 and 1306 and S. uvamm R2 and NCYC 1326, 1324 and 1343 were 0.33, 0.39, 0.25, 0.71,0.33 and 0.51 ng DMS formed rain °~ mg pro. tein "1 respectively.

3.2, Partial purification o f yeast DMSO reductase

Preliminary experiments suggested that DMSO reductase of yeast might be identical with MetSO

$7

reductase which involves thioredoxin and thioredoxin reductase [20] and thus initial attempts to purify the proteins involved followed methods used for this system. Fraction A was prepared by the method described for "Enzyme lIP' by Black et al. {14] with the omission of the f'ma] adsorption stage. Fraction B 't~aSMpUrifi~ ~ described for thiorcdoxin and tldore- doxin reductase elsewhere [21 ], finishing at the heat stage. The heated supematant was taken to 90'~ saturation with (NH4hSO4 at 4°C and the precipi. tale 1edissolved in tl~e minimum volume of 0.2 M phosphate, pH 7.0. It was dialyzed overnt~t at 4"(2 against 50 voL of 30 mM phosphate, pH 7.0. The sample was then applied at room temperature to a column (1 cm × 4 era) of DEAE-cellulos¢ (Whatman DE-52) equilibrated in this buffer. The column was washed with 3 vol. each of 20 raM, 50 mM and ! !0 mM phosphate, pH 7.0. Those fractions eluted at the highest phosphate concentration were pooled and stored at - I 5*C.

Fractions A and B separately showed little DMSO reductas¢ activity whereas their combination stimu. lated activity (Table I). Both fractions were also necessary for the reconstitution of MetSO reduetase, suggestLng that both reactions are catalyzed by the same enzyme system in yeast.

Measurement of MetSO4ependent NADPH oxida- tion was often masked completely by the oxidation of NADPH catalyzed by fraction B in the absence of added MetSO. The endogenous activity was pre- cipitated by (NH4)aSO4 and was non-didyzable and is presumably, at least in part, a function of the presence of thioredoxin and thioredoxin reductase in this fraction. Chromatography of fraction B on Sephadex G50, which had been previot~y found to

TABLE 1

Reeonstitutioa of DMSO reductas¢ and Met$O reductase

Enzyme tractions A and B were prepared aM assayed as described in the text, Assays contained 770 ~g protein A;46 t*g p~tetn B.

Fractions assayed DMSO reductase MetSO redueta~ (rig DMS formed rain-! ) (nmol NADPii oxidized min "z)

A 0,05 0 B 0.06 0 A + B 0,28 2.9

58

resolve these proteins [21,22], did not separate them hi this instance, as shown by the retentibn of DTNB reductase activity in rite same fractions. Such frac- tions did restore DMSO reductase activity to fraction A whicl~ had been further purified by addition of calcium phosphate gel,

3.3. Further evMence for rite im,olvement o/' the thioredoxin s),stem in ),east DAISO reductase

Thioredoxin reductase and thioredoxin are involved in providing reducing equivalents for the yeast enzymes ribonucleotide reductase (EC 1.17.4,1 ) [19], sulphate reductase (EC 1.8.1 ..) [20] and possibly sul- phite reductase (EC 1.8.1.2) [23]. As expected, sub. strates for these enzymes interfere with DMSO reduc- tion, presumably by their competition for reduced thioredoxin (Fig. 2), The true substrate for sulphate rcductase is 3'.phosphoadenosine-S'.phosphosulphatc (PAPS) which was not tested in the presellt system. Sulphate can be converted to PAPS by crude extracts of yeast [24] and it is assumed that this is the inhibitory factor. NASH had little effect on DMSO reduction. Oxidized glu~athione (GSSG)stimulated DMSO reductase in crude extracts by 2.1-fold at 1.8 mM and 2.7-tbld at 7,2 raM. Such activation may reflect a role for the NADPH-lirtked enzyme gtuta- thione reductase (EC t.6.4.2) in DMSO ,eduction, This enzyme can reduce thioredoxin in L" coil [18].

3.4. DMSO reduction in bacteria

Extracts of beer.spoilage organisms E. coli, Ent. cloacae and F. proteus catalyzed NADH- and NADPH.

o.a ,

/

/

O.

i I , J . ~ il ! ,, ,,, ~ [

o I 2 3 4 5 6 ?

InNbito¢ ¢oe~onlratlon(m~

I #. 2. Inhibition of DMSO Reduclase activity in crude . ,reacts of $¢tccharqmyces cererlsiae (NCYC 240), Tile .~tandard assay was used, including tnhibitots as shown with- out prior iacabation: %cy~idinc 5'-diphosphate (CDP); o, potassium sulphate; e, polassium salphite; =, NADH, Enzyme source was a crude extracl ofS, cerevisiae (NCYC 2401,2.7 mg protein.

linked DM$O reduction (Table 2). NADH is the preferred electron donor. NADH.linked activity was inlfibited by 5% at 7.2 mM CDP which caused 15% inhibition of NADPH.linked activity. A crude extract of Era. cloacae was centrifuged at I50000Xg for 1 h. The soluble fraction reduced DMSO in the presence of NADH at a rate of 3.9 ng DMS formed rain -t mg protein -1. The membrane fraction showed a rate of 4.5 ng rain "t mg protein "t. Combined, the rate was i0.4 ng rain -1 mg protein -1,

TABLE 2 DMSO re.ductasc in bacteria

Extracts were prepared and assayed as described in text, using t rag extract protein.

Extract Electron donor DMSO reductase (ng DMSO formed rain -I mg protein -l)

Escherfch~a ¢oli NADH 2A1 NADPH 0.53

Enterob¢cter clo¢¢¢t NADH 8.2 NADPH 1,0

Flavobacterium proteus NADH 2.93 NADPH 2.07

~9

Discussion References

The evidence suggests that DMSO is reduced by different enzyme systems in yeast and bacteria. Yeast enzymes did not use NADH to reduce DMSO whilst NADH is more effective than NADPH with bacterial extracts. It seems that DMSO reduction by yeast is catalyzed by an enzyme similar to, if not identical with, MetSO reductase. It has previously been shown that MetSO reductase involves thioredoxin and thio. redoxin reductase, an electron transfer system involved in channelling electrons specifically from NADPH to a range ofacceptors [25]. The system is found in yeast [21|, bacteria [26|, blue-green algae [27] and liver [21 ]. The thioredoxin system in E colt is NADPH-specific and so DMSO reduction in bacteria cannot be entirely catalyzed by MetSO reductase. MetSO reductase of E. cell has been shown to be similar to the yeast enzyme except for its apparent division between soluble and particulate fractions [15]. It seems that a similar subcellular division ob- tains for the NADH-linked DMSO reductase of Ent. cloacae.

In contrast to DMSO, BioSO is unlikely to serve as a substrate for MetSO reductase of yeast. The finding that BioSO is not an inhibitor of yeast DMSO reduc- tase agrees with the observation of Dykhuizen that mutants of E. eoli unable to reduce BioSO could reduce MetSO [28]. It is likely that MetSO is the major substrate for the present enzyme as it has a much higher affinity for the enzyme than DMSO [4] and also as MetSO is reduced at approx 640 times the rate of DMSO (Table 1). It should be noted that previous workers could not demonstrate DMSO reduction by MetSO reductase [14].

Whereas the role for sulphoxide reductase in yeast is probably to maintain the respective sulphides in the reduced form [11,14], NADH-linked DMSO reductase in bacteria may serve as a terminal dectron accepter system for anaerobic growth [10,29], of. trimethyl- amine N-oxide [30].

Acknowledgment

Mr. H.M. Dunn is ~anked for technical assistance.

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