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l,tnu.~r> 19UI

Identification of sulfurtransferase enzymes in A:.otobacter vinehtmfit % P,tg,mi =, E. l-t,mch= t, R C'oln,tghl ~ and C', Kcnnedy = I)#t)(trt#t)#¢/H,) (11 .S¢ t¢~1".¢ Mr,let ()htr# 4groahnt¢ttt¢lr~ (#u/ ('entre /tllerltttlt ¢ri#l(trlo I)¢r Io ,'~'llaho de/It" A~(I( roltN)l¢¢ to" ht/ortthl:tUthth t 'ms rr~ttl of ,Ihhtn. h(t/t a n d ". | k R(' h~st)ttttc ¢11 P/ant S, refit ¢ R¢~c~lrt h. ,%'ttr.gcn h x~ltt¢)tt lathorat.r) Ilrlghton. fl,VI o p Q . L'A l~,c~.cI~,cd22 No~ el'riDer IqgO Rh,)dalleS¢ altd t 111cr,.aptopWtl~atc suli+hutll,lllSlcr,~sc h.t~c bCcli idenlilted lit ~.~ +meholdo r~Ao dlMin,,.[ .t¢,,{l~,e rr,l~.llOllm I)f the ts~,o sulpiturlr.~llS fer,~sc.t ~ere ,)bt.ltncd ,if let [ PI ( mn e~.llall~c ~.hroln.~tugl.ll)h.~, o f in,ltcrl.tl p irt=.dl) purified front crude cxtr,l~.ls I~.hodancsc has I~cl'l pUrlhcd 1o ho)t~ogcncits..itld 11 t.o(}sisls t.f one poisl~pude ~.h.lln ur ,i,f~ ~.,i 25o1~) A p,lltl,tl purih~.,ttlo~t of '4nlcr~.,iptop.~,rus.ttc sulpiturtr,tnsfer.lse w i~, t ~bl,i i i'lc~ I

4.olul)lt~/re leith#lob,, 5U',l)ltuth,lllslct.~sc. I>.llt)d,lllcsc. "t,b,icr~.,iptop~,ru~,lte sulphurtt.Wislcr.isc

1 INTRODUCTION

2 MATERIALS

The sulphurtransferase ,'hodancsc (Rd) (tluosulphate-cyan~dc ~ulphurtr,lnsfera~e EC 2.8.1,1) and 3-mcrcaptopyruvatc sulphurtran~ferasc (3-MST) (EC 2,8,1,2) catalytc the transfer of a ~ulphane sulphur atom from a donor molecule to a tluophdtc accepter substrate [I-4] Although wldcly d~strtbuted, the physiological role of these enzymes has not been estabhshed [1,5] Prev:ous work demonstrated that m a m m a h a n Rd ~s effective m the m v~tro synthesis of heterometalhc clusters [6-9], In addition, macnve Feproteln of the mtrogenase from Kleb~te/la pneumomae, lacking the full complement of ~ron and sulphide, could be rcacnvated for acetylene reductlon after incubat~on w~th Rd and its substrates [10]. Low levels of Rd and 3-MST were detected m cultures of K pneumomae [11]. In prehmmary experiments, actlv~nes in Azotobacter vmelandn, another nttrogen fixmg orgamsm, were more stable and ten times higher for Rd The aim of the work presented here was to verify whether the sulphurtransferase acuvltles detected m A vmelandn extracts corresponded to specific endogenous enzymes. Sulphurtransferase enzymes generally show 1o~ substrate specificity [5], and recently A~rd et al. purified from the prokaryote Acmetobacter calcoacettcus Iwofft a sulphurtransferase which utihzed a w~de range o f accepter substrates [12]. We report here the identff~canon and purificanon of Rd and 3-MST from cultures of A. vmelandn

.d.~otoba, trr tmeh#mllt strmn Ll',A'136 (from NFL ¢olle~.[tol'0 ~,as grm~n in Burk's ~u,.ro~e Inedlunl [I 3] irt .t 200 htrc Ne~ lJrult.s,,,,tck st,IHllcss '~tccl fermento) AcrdtlOll rate t~a'. 50 I m=n t Ceils %%ere harvested m a Westphaha ~.eatrlfu/ie whel3 the culture rc,tchcd OD,~-7 2 0 E'stracls ~¢re prodm.ed bl, tha~,,a~g50 g of fro~,en ~.cll~in 75 ml of 50 m M phosphate buffer ~.ontamln8 3 5 m M thlosulph,tte (pH 7 O) rhc suspension ~as passed t,~ ~e t hrou/~h a Cell l'rm.tlOlmtor at 28 psi el ,ugon Cell debris v,,t,, relllo',ed by ~.¢lttrlfugauoll at 1.3 LX)0 x g for 60 ram, the supernatant s~as used for the purification of sulphurtrdnsferd,,es Proteins ~erc cstmtatcd caher by the blurct method [14] or m the ~.,tseof parthdly purified material, from the absorbanc¢ at 280 n m by using the cxtmctlon coefflctcm of l 50 for a solution containing I 0 mg/ml Rhodancse acn~.Ity ~as dctelmlaed by the colorlm,'tri~,assa!, based on the absorption at 460 nm of the ferric tluocyanatc complex formed from the reaction product th~ocyanate and ferric nitrate [15] The m. tlvlt!,, l,gure~ v, ere corrected for the absorbanc¢ at 460 nm of a parallel assa}, m s~htch tl~e sample v,as added after the addltton of H C H O m the cubat,on mixture The 3-mercaptopyruvate sulphurtransferase a~say ~s ba~ed on the detecnon of the product pyruvate [4,16] The ab sorbance figures were corrected by subtracting the value obtained when the en,,yme preparation was added after CdDI~ One unit o f Rd and 3 MST actw,ty is defined as the amount of enz.~,me ti~at produce 1 ~mol o f d~locyanate or pyruvate per mm at 370C

AND M E T H O D S

2 I Purtfwanon o f the sulphurtrans.ferases 2 1 1 Step 1 A m m o n , u m ~alphat¢ precipitation The extract ~,as made 65% saturated w~th ammonium sulphate A f t e r centrltugauon at 13 0 0 0 × £ for 30 ram, the preclp,tate was red,s~olved m 50 m M phosphate buffer, pH 7 0, eonta,nm8 3 5 mM thlosulphate (buffer A) Unless otherv, lse stated, all procedures were carried out at 4°C 212

address S Pagam, D=parumento d= Sclcnze Molecolan Agroahmentari, Via C e l o n a 2, 20133 Mdano, Italy

Step 2 Sephadex GS0 fractlonatlon and ~on-exchange chromatography The red~s~ob,ed proteins from Step l (50 ml) were fracttonated on a column of Sephade× G S 0 Fine (Pharmacm, 7 5 × 40 cm), equ,hbrated with buffer A,. t a flow rate of 200 ml/h The fractions with high salphurtran~ferase actl~,mes were pooled and passed through a

Pubhshed by Elsevter Sctence Pubhshers B V (Btontedwal Dtvtston)

151

Correspondence

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~ o l u n m el Ill AI %eph,t~.cl (V, h,ttm,m, ~ ~. ; ~.m)equdtbr,tted tn but l e r X %tnlphtJrlr |ll~I¢l,|~t. dt. ll%l|le~, ~t.r~. re~.o~cr¢d It| t h e IlOll |lt'~tll~tl Ird~.llOlI l h c ,l~.IP, e Ill,|lt. Fldl LltltCd t r o t h t h e I)1 AI %qth.t~cl t.oltlllll~ ~.P,

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sulph tie (6~°o) dt pll 7 t} Alter ~.entr,lug,tliu,I tl~e pl~.~.,pzl.tt¢ ~,t~ rcth~,,ol~td in 5 ml of It) raM phosph,tt¢ b u l r c r pII "~ O, ~.utlt,ti,nt~g ~1 In%l Ihio~tJIl~h,itt. ( I t t i f l c r I~) ..2 I "~ %top a l.)cs,dttng and I Ill ( ion c\~.ll,|llgc ~.hroln,ttt~gr.q~h~ br,ttt.tl ~Ailh but h.r l} I he clu,|te ~.|., Oh |deal m dht|uot~ of 2-2 5 l|~itl p r l l t e l n ',~,hl*,.It ~t.'re ,~t2i'~tr,ne[~, I o , I d c d O11 ,t T%I%-I)[ A I 51)%t,' t.Oltll|'lll ([ kl}, 0 8 × 7 5 ,.hi) e q u t h b r a t c d m bt~tfer I I m i 1 PI (. ,tpp,tr,ittts (1 1%1}} ,it fOUll'i tClUl~er,~turc Llutton el bOtllltl proteln~ ~.t', at.h~e~¢d b) ,t htle,t, gradient el b u l l e r [i to h u l l e r A it1 25 111111( | II,~ I ) lhl~ ~tep e'~,elllldll '~ ~ t p d r , | t c d Rd

iiId 3 %1~1" .tt.tl'~ltl¢ ~,

2 I 4 %I~p4 (Jd h l t r , t u o n b~ I I ' I . C I hc ~.ot~lbined m,ttcl ill ~ tth Rd ,t~.h~lt~ f r o m three I PI C - D L A I eltlttOIl', (pc,tk II el I Ig 1) ~ , h ~.ont.t.llll tied ,.tbt~ut '~0-1old b~, ttmng

li.FIFR%

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It)91

,I (~ eVIl II~, on I o {AIIII~.OII) tlt.~, h.~,, a n d I o , t d c d oil ,I ~,t~ltll|lll tit %tll'JCfO~¢ 12 It) lO IlK (I Nil. I -. 3U,,.m}t.quihhfdtcd ,tl ~t),nM phosph.tlc huf f,,,[ ,,.tltlt,tih|l~[.' ( I I XI N , t ( I ( p l l " ") in I P L C .Ipl~ Ir.tttt~ Rd x~ t', ,.,luted V, t l h t h e ,, t11|¢ b t l | l C l ,it d l i a r , i,l|,., ill (] ~ II*11 II1111 I he '~.n||c [ll~,l~.cl.[tlrc ~1,d~, toIlo~'.'.'.l Int ll~¢ p'. tk ~ t~l I lg I ~,.Ol~|,|il|lll]: ~ C'~'~¢ltll li[~

"~ %1%1

3, R F S U I . T S A N D I)I%CUS'51ON Rhod,tne~e

3.alert.apt Ol~yt uvate ~ulphut i n Cl t i d e extrac.t~ at A t'ltle/atldlt grox~n ~ tth etthct 15 mM amnlonltml acetate (spec act at 0 1.12 and 0,183, respectively, lot Rd and 3-MSF) el dtlnttogen (~pet a~t, at 0 300 and 0,410, tcspoctt~,,ely) ,'1',nitrogen ,,otn c0 Tile actt,.,ltien v.cte only ~hghtly higher Ill tile dlnlttogen-grox~n cells, ~uggesttng Ill,at neither was ,t product of a nitrogen lixatlon~pet.tttc gene, Snlce sull~hurtran,,lerase activities in tile presence o f dt t lerent d o n o r molecules are not necessarily ~pectltc t a r the identthcatton o f t ~ o ~eparate cn/~mes [5,12], we ,tttempted the purtlicatton at tile Rd and 3-MST activities tran'dol

and

,ls¢ a c t l V l t l e ~ ~ Cl e f o u n d

3 1 Pu.ftcatton of the enzymes from N.,-grown cells The scheme for purification ts shown m Table I Durmg tile early stages, both activities were recovered m tile same fractions In order to achieve separation on F P L C ton-exchange c h r o m a t o g r a p h y ° we round tt necessary to first remove bulky contaminating proteins on a Sephadex GS0 column, although this led to significant loss o f materml. Afte, FPLC c h r o m a t o g r a p h y , two distract peaks were recovered (Fig 1) The fraction carresponding to 3-MST contained 3.307,0 o f residual Rd a c u w t y The peak containing Rd activity eluted lust behind that of 3-MST, m the Rd fracuon the conlaminating 3-MST accounted for 5070 of the total sulphurtransferase a c u v m e s . Further purification o f the two enzymes was achieved by F P L C gel c h r o m a t o g r a p h y on Superose 12HR (Table I), Both enzymes were, at this final stage, purified m o r e than 1000-fold c o m p a r e d to the crude extract specific activities and no cross-contaminating a c t i v i t i e s were f o u n d (Table I),

0~0-

E~

I E 0 C5

D

3.2. Homogeneity and molecular weight o f the purified enzymes

0 00I 20

I ,40

I 60

Fig 1 C h r o m a t o g r a p h [ ~ s e p a r a t i o n o f Rd and 3-MST A typical c h r o m a t o g r a m o b t a i n e d with a h q u o t s o f m a t e r m l f r o m S e p h a d e x G25 pas~ed t h r o u g h the T S K - D E A E 5 P W F P L C c o l u m n ts s h o w n A a n d 13 are peaks of 3 - M S T a n d Rd activities, respectively

152

When the 3-MST fraction f r o m F P L C - D E A E was c h r o m a t o g r a p h e d on Superose, two m a j o r and several r u m o r peal~s were obtained (not shown). The 3-MST actw]ty was found only m one peak which eluted at a position corresponding to an apparent Mr or 25-26 kDa. This peak gave two m a j o r polypeptlde bands (Fig 2) when analyzed by S D S - P A G E that accounted for 61070 a n d 33°70 of the total proteins loaded. Their a p p a r e n t Mrs correspond to 38 and 29 kDa, respectively Since we did not succeed in the purification of 3-MST to h o m o g e n e i t y , the deftmtton of its molecular weight req m r e s further investigation

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Atter ch~on~atog)aphy on Supeto~e ot the Rd traction tlona IrpLC-DEAE one single peak with an apparent Mr of 24 kDa wa~ found (not shown) SD$P A G E ot tl~e Rd eluted trom Superose furthel demonstrated the p~e,.ence of a ~mgle polypcptlde species (Fig, 2) with an appalcnt Al, of 29 kDa. Moleculal weights ot the same o~der of magmtude as that of naammahan Rd (33 kDa) [17] and also of less than 20 kDa have been rcpmted for Rd's ~solated from prokaryotlc orgamsms. Th~osulphate-uuhsmg bacteria

431

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contain Rd% ~ith s1,'es ranging from 34 to 39 kDa [18,19]. Rd ttom F.schertchta tub, on the other hand, had an Mr close to 14 kDa [20] Rd from the soft bacterium Acmetobacter calcoacettcus showed a Mr of around 35 kDa [21]; also recently Alrd et al, [12] purified lrom the same bacterium a sulphane sulphurtransfelase w~th an apparent Mr o f 17 kDa, Thus the rhodanese purified here lrom A, vtnelvncht falls somewhat midway m size between the two extremes of molecular sizes reported for this enzyme. Enzymes belonging to the class of sulphur transferases utilize or produce sulphane compounds [1,5,22] The physiological function of sulphane sulphul, however, has not been well defined [23]. It could be the source of the morgamc sulphur used for synthesis of iron-sulphur centres. Though the enzyrmc system based on rhodanese has been extenslvely utilized tn the m vitro synthesis of metal-sulphur structures [6-9], It remains unclear whether this enzyme can be regarded as the m vwo catalyst of reactions revolved m the synthesis of metal-sulphur structures [24]. The Identification of two different sulphurtransferases in A vmeland., as well as the purification of rhodanese to holnogenmty, represent an important first step to ~nvesugate the role of these enzymes by combined biochemical-genetic approaches ,4cAnot, ledgements We ~slsh to thank Dr g R Ead~ for helpful

D

¢

d

e

Fig 2 Electrophoretle analys~ o f the final enzyme preparatmn SDSP A G E ,.'.ab performed according to Laemmh [25] Samples v, ere denatured m the presence o f I 25% 2-mereaptoethanol and 2~0 SDS (Lane a) Rd actn.e peak Irom the TSK-DEAE 5PW column (15 t.,g), (lane b) protein markers (15 ~,g total of a m:~ture containing phosphorylase b, bovine serum albumin, egg albumin, carbomc anhydrase, trypsin mhzbttor, a-lactalbumm, and a p r o t m m ) (Lane c) Rd from the $uperose 12HR column (10 t,g), (lane d) 3.MS1 aetr,'e peak lrom the TSK-DEAE 5PW column (20 ~g), (lane e) 3-MST actr.e peak from the Superose 12HR column (10 .ug)

d~scusslon. F Ses~a for techmca] asslstance. R Humphrey for gro~,.m g large scale cultures, and Prof B E Smith for construcuse cc~mment~ on the manuscript The present m~.estlgat)on ~sas supported by g r a n b from CNR (Itab) C T 88 02275 06115 060"]3 and C T 89 04895 06 I15 060"]3

REFERENCES [I] Westlet,, .] (197 '~) in Bloorgamc Cheml~try. vol I (Van Tamelen, E E , ed ) pp 371-390. Academic Pre~s. Orlando [2] Vfl]arejo. M and Westley. J (1963) J Blol Chem 238. 4016-4020

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[lloph)s

[.~] Vie,ltlcy, J (19141i)III Eli,t~iil.illt. II,i~i,, ~lf D¢lO\il'lv,lliuil, ~,(ll 2 (J,It, ol'l>, W I1 cd ) pp 245-261 [6) liagtllll0 % , ItolIO~til, I dlid (. i.rlcttl, I ) (I,)1~,1) I ui J Ihm.hciil 141, 361 -,166 171 IlolIOlili, t , P,ill,llll, ~ ,llld Ix~lrl.~, I . ) M (19i45) Lur .I Iliocheil'i 141t, 67-GI3 I~1 liOnOIlll, I ~ , Wcrlh, M i ,li,ld IxLirl#, I) ,%,1 (lt,)l~l! I) IllOl'bl ( Iic111 24, 4311-4335 [9] IIOl~Oilii, l , lanlelil, 5 , kill-It, I) M .ilid P,il~ani, ~ (19t'18) ,11 Nltl'Ollcn I~l%,"iilOll tiull(lretl ~"¢,Irs ,&flcr (ilolh¢, I'i , dc ltruull, ! J , Nc~,,ioi'i, V¢ L cds) I~ 6:~ IIO1 l)~t41illi, S , Eldi~dS¢, ,'%1 ,ind [ ,ltl',, I{ it (1987) lJio~.li~iil .I 244, 4~5-4~8 [I I] lillg,.llll, 5 , BOilOllll, I~" ,lllt, I k e n n e d ) , C (198B) lho~.liclll (LII+,.' S~.i e'lld~ )7, 27~i-2itl [12] Aird, II A , Helnrik~on, R l ,ind W~s/ll.y, J (19'17) J Ihol C'll¢ln 262, 17"~27-17335 {13} N c ~ i o n , J W , Wd~on. P %%' .rod llurri~, R FI (1953) J Biol Cll~.nl 2fl4, 445-451

154

L r'm'ra,,

1.1:~° 1-16 II'/I l)lo¢~-nmIl, J I I , Droll| (,, I~o111~.I Ix l l , tIul, ~,~. t . , . I , I l c m r l k ' , m h I,L L , L)¢.ll P . WCn~, I .Lfld Hu~clI, J (l~.~?x) Naliire 27~, 124~12L,~ [ I x ] IruPct, II t., (I'~xl) in Ih~flot]}, o f Itlor~.iul~. Nilrt~gcn .Ind '~ulphuf (I]oth¢, tl and rlCb~.l, A Cds ) DP ILJg=21 l, .%pr,nger, Ltcrllll lliJi SlCilllllCi,~, b,! ,.'% ,llld l i,,hcl, 13 (198~i) Ar,.,h M l l r O b i o l 14-"l, [20J Alcx,uldcr, k , Vuhlli, ~,l (I,)1~7)J Ihol (r'hClu %2, 65,)5-6604 [2lJ V,lild¢ilbert41l, P A lind Ilerk R S (19hO) (.,lit J Mi~.rOhIOI 26, 2111-21~6 [22] Ro>, A I,I .illll ] rtlthlll.l¢l, R ~t (197()) m I lie lhochen'ustr~, ot IllOrl~dlli- (.Oflll'lOilnd', Of ~llll'~lll, if, (,.illlblidLtc UIII~, l~'rc~,,. C nlllbrldgc UI~ 57~5-5740 [24J Ccrlctti, I ) (1986) lrends Iho~.heill 5~.i 11, 369-"47: [:$1 i ,iCltllllh, U Ix (1970) N,ilUl~. 2.'!.7, 6t40-614f

Identification of sulfurtransferase enzymes in Azotobacter vinelandii.

Rhodanese and 3-mercaptopyruvate sulphurtransferase have been identified in A. vinelandii. Two distinct active fractions of the two sulphur transferas...
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