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Ribitol-5-phosphate dehydrogenase from Lactobacillus plantarum
BIOGHIIgICA I~T BIOPHY.'XIt~A AC~A,
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BBA xzz93 RIBITOL-5-FHOSPHATE DEHYDROGENASE F R O M L A C T O B A C I L L U.q P L A N T A R U ~ I LUIS GLASER
Da'partr~=l of Biological Ckem~try. Wash~n~'~. Uni~er_~,ly School of ,~qr~i¢i,w. .S'ai~t Louis. Me+ (U.S.A./ ( R e c e i v e d J u l y tGth. I S Z j
S U M M A I~a(
T h e partial purification o f a r i b i t o L s - p h o s p h a t e d e h y d r o g e n a t e from Lactob.~cill,,s plant.arm (ATCC 8oi4) is described. The e n z y m e has linen shown to catalyze the reaction TPN ~ TPNH r~--~bitol 3-phcmphe~t~ + or ,# D-ribu[o~e 5-|>hosph~,t,: + or + ]{+ DPN ~
DPNH
INTRODUCTION
I n 1954 BADDILEY ^~D MATHIASx described the isolation of COP-ribitol and later the presence o f ribitol teichoic acids (a family of sub~titutexi polyribito! phosphates) in a v a r i e t y of gram-positive b a c t e r i # . T h e enzymic svnthesis of CDP-ribitol from D-ribitol-5-P a n d C T P has been described b y SHAWa. No e n z y m ~ which specifically catalyze t h e synthesis of D-ribitol-5-P have been isolated although an a p p a r e n t l y tmspecific aldose reductase from silkworm blood will catalyze the reduction of or i b o s e - 5 - P to D-ribitol-5-P (see ref. 4)- W e wish to report the partial purification of a n e n z y m e which c a t a l ) ~ s the formation oi ~ b i t o l - 5 - P from D-ribulose.3-P, using either D P N H or T P N H as a h y d r o g e n donor. EXPERIMENTAL
Mataria2s Laa0bact'Uus #Lantarum (ATCC 8o14) was g r o w n in a m e d i u m oQntalning per 1: 4 g o f Difco y e a s t extract, xo g o f Difco nutrient broth, ro g of r ~ l i u m acetate. o.a g o f MgSO,. 7 H a O , o.ox g o f NaCI, o.o! g of Fea(SOi)s. 9 HtO, o.oz g o f MnSOa" 7 H t O a n d ao g o f glucose. An m o c u i u m from a ~iab w ~ g~o'~.~ in Too nfl of this medimm~for xa h, a n d 6 ml -,f this c u l t u r e were used to inoculate 3 ! o f m e d i u m in a 6-! E r l e n m z y e r flask a n d t h e culture allowed to grow for xz h at 37 °. The ceils from x8 1 o f c u l t u r e m e d i u m were bin-vested b y centrifugaGnn a n d w a s h e d tw~:ce with 4oo nfl of o.o5 M T~'~-HCl~o.oot M E D T A ( p i t 8.o). The ceEs were fmaUy suspended in a mininml v o l u m e o f o.o5 M Tris~HCl-o.ox M MgCtt-o.ooz M E D T A ( p H 6-0) ~ d Biockim. Bio#t~5. Aaa. 67 ~1963) 5 ~ 5 - 5 3 o
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lyophilizcd. The lyoph'"-flized powder could be stored for at least 3 weeks at ~ 2 o ° without loss of activity. ~ . R i b u l o ~ - 5 - P was prepared b y the m e t h o d of Hom~cKat~'. The 6-phosphogluconic acid d e h y d r o g e n a s e s used contained ritxJ~-5-P isomcrase and the final product, even after column c h r o m a t o g r a p h y , was c o n t a m i n a t e d with some ribose-5-P. Pentose was d e t e r m i n e d b y the orcinol m e t h o d e, with a 4o-min heating period in a volume of r.5 ml. Ribulose was determined b y the cysteine carbazole reaction ~ using a u t h e n t i c r i b d o s e as a standard. R i b u l o s e - 5 - P was d e t e r m i n e d b y the same m e t h o d after r e m o v a l of the p h o s p h a t e with chromatographically purified E-chcrichia coil phosphatase (Wowthington Biochemical Corporation), thus permitting the use of free ribulose as a s t a n d a r d ~n ille colorimetric assay. Aldopenta.~es were d e t e r m i n e d b y the phloroglucittol m e t h o d n. t~-Ribnh~e-o-nitrophenylhydrazone was obtainc~l from California Corporation for Bi~ychc-m~cal Re.~arch. Ribose-5-F from S c h w a r t z Laboratories, D P N H a n d T P N H were obtained from the Sigma Chemical Company, R i b i t o l - f - P was p r e p a r e d b y the methtxl of BADDILEY tit a / , 9. R i b o s e - 5 - P isomerase wa_~ d e t e r m i n e d b y incubating enzyme with ribose-5-P (see ref. to) under the conditions of the ribitol-5-P d e h y d r o g e n a . ~ assay. At t h e end of ineuV:ation, the sugar p h o s p h a t e s were h y d r o l y z e d with E. coli p h o s p h a t a ~ and the ribulose formed determinedL Protein was d e t e r m i n e d b y the m e t h o d o f WAI~BUaC; :_XD CXmSTl^N n. P r o t a m i n e sulfate was obtained from Si~,ma Chemical Company. Fresh solutions of p r o t a m i n e sulfate were prepared for the e n z y m e fractior~ti_~n and neutralized to p H 7.o with K O H .
As.say of ribitol-5-P dehydrogenase The reaction ntixture contained: t o # m o l e s of T n s - H C I , o.2 l,mole o f E D T A , 0.3/1mole of ribulo.se-5-P , o.x/~mole of T P N H and 500/~moles of 0anmonium a c e t a t e in a final v o l u m e of x ml {pH 7.8). Control reaction m i x t u r e s c o n t a i n e d 811 t h e c o m p o n e n t s minus ribulose-5-P. A b ~ r b a n c y m e a s u r e m e n t s were t~keP at 340 m/~ ever 3' m i n u t e in a B e c k m a n s p e c t r o p h o t o m e t e r t h e r m o s t a t e d at z5 °. I n all the e n z w n e fractions except the final p H - 5 precipitate (see below) ribose-5-P could s u b s t i t u t e for ribulose-5-P w i t h o u t change of activity. The e n z y m e could also be assayed b y measuring T P N H formation from ribitol5 - P and T P N . The reaction m i x t u r e c o n t a i n e d x o / a n o l e s of Tris--HCl, o.~ p~nole o f E D T A , z #moles of ribitol-5-P, 5 / l m o l e s of T P N and 5 o o ~ m o l e s of a m m o n i u m a c e t a t e in a final volume of I ml (pH 7.8). W i t h the crude extract, values o b t a i n e d b y this &ssay are low due to the presence of T P N H oxidase. I unit is defined as t h e a m o u n t of e n z y m e required t o catalyze a change of o.ox a b s o r b a n c y unit/rain. T h e d e p e n d e n c e of the a c t i v i t y on e n z y m e concentration is shown in Fig. z. RESU LT~
PHrifu:ation of ribitol-5-P dehydrogcnas¢" x g of d r y cells was suspended in 8 ml of o.o5 M Tris-HC1, o.oz M MgC11. o.oox M • All s t e p s i n t h e e n z y m e p u r i f i c a t i o n w e r e c a r r i e d o u t a t 3 °. A• p r e c i p i t a t o r s w e r e c o l l e c t e d b y c e n t r t f u g a t i o n a t z2 c o o × g f o r t o r a i n . A m m o n i u m s u l f a t e w a s ~atur~Lt~t a t 3 ° a n d n e u ~ z e d w i t h . N ' H . O H s o t h a t a I : 5 d i l u t i o n h a d a p H c~f 7.5-
Biockim. Biophys. A~:ta, (17 0903) 5~5-~$o
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Fig. t. t)cpcndencc of ribitol-5-P dehydrogena~v activity on enzyme c~,,centration. Stand~r0 assay conditions; tit.: enzyme was ammonium :~ulphatc ftatt:tion. E D T A ( p H 8.o), s h a k e n w i t h 8 g o f glass h e a d s in a Nossal s h a k e r for t h r e e 3n-see p e r i o d s b e i n g c o o l e d in b e t w e e n e a c h 3o-.~ec p e r i o d in a n ice b a t h . T o p r e v e n t fc~'aming. a d r o p o f c a p r y l a l c o h o l w a s a d d e d . T h e gla_~s h e a d s w e r e allowed to s e t t l e a n d t h e s a i p e r n a t a n t fluid c e n t r i f u g e d . T h e b e a d s a n d p r e c i p i t a t e w e r e wa_~hed t w i c e w i t h 5 m l o f t h e s a m e buffer. T h e p o o l e d s u p e r n a t a n t fluid was t h e n f r a c t i o n a t e d w i t h p r o t a m m e sulfate. In a t y p i c a l e x p e r i m e n t as show.~ in T a b l e I. I8. 5 ml o f 2 % p r o t a r n i n e s u l f a t e was a d d e d t o t h e e n z y m e ar.d a f t e r in rag,, t h e p r e c i p i t a t e wa* c o l l e c t e d b y ce,Lt~ifugali,,, T AI3LE l .
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a n d d i s c a r d e d . T o t h e s u p e r n a t a n t fluid a n a d d i t i o n a l i 6 m l o f 2 % p r o t a m i r ~ s u l f a t e w e r e ad.:~-,d a n d a f t e r I n rain t h e p r e c i p i t a t e w,~s c o l l e c t e d b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a . l t fluid d i s c a r d e d . T h e p r e c i p i t a t e f r o m t h e s e c o n d p r o t a m i n e a d d i t i o n was w a s h e d w i t h 2o ml o.~ M a m m o n i u m a c e t a t e ( p H 7.8) a n d t h e n e x t r a c t e d for 0. 5 h w i t h z5 m | o f x M a m m o n i m . , a c e t a t e ( p H 7.8). T h e i n s o l u b l e r e s k l u e was r e m o v e d b y c e n t r i f u g a t i o n a n d d i s c a r d e d . T o t h e s u p e r n a t a n t fluid, c o n t a i n i n g t h e e n z y m e , w a s a d d e d a n e q u a l v o l u m e o f s a t d . a m m o n i u m sulfate, a n d a f t e r iS rain t h e precib,~:.~- w a s colle."t 1 b y c e n t r i f u g a t i o n a n d d i s s o l v e d in x M a m m o n i u m a c e t a t e ( p H 7.8j. T h e , ' - t z y m e w a s t h e n d i a l y z e d a g a i n s t o . I 5 M a c e t a t e b u f f e r ( p H 5.x) w i t h
Bio~Mm. Iiiop#tys. AcM. 67 (x963) 525-53 °
5Z~
L, GLASER
stirring for 4 h. FinaLly the precipitate was collected b y centrifugation a n d dis.~olved in ~ M a m m o n i u m acetate (pH 7,8). The e n z y m e can be k e p t frozen at all stages in t h e purification for at least a week w i t h o u t loss o f activity. The s u m m a r y o f a typical p r e p a r a t i o n is s h o w n in Table I. I t has been folma necessary to pilot the p r o t a m i n e step w i t h ever3- e n z y m e preparation, siuce ~.b.z quanL~ty o f p r o t a m i n e which will precipitate m~x~t o f t h e nucleic acid b u t not the e n z y m e , varies from p r e p a r a t i o n to preparation. After the p r o t a m i n e step the e n z y m e becomes u n s t a b l e at low ionic strength° a n d dialysis of this fraction against o,z M p h o s p h a t e (pH 7.5) or o.z M Tris (pH 7-5) for 4 h leads to complete loss of acti-rity. Precipitation at p H 5.1 causes a relatively large loss of a c t i v i t y b u t serves to r e m o v e m o s t of the vibose-3-P isomerase. Reprecipitation o f the e n z y m e a t p H 5.z does not remove the last traces o f isomerase. In t h e p r e p a r a t i o n shown in Table l, the e n z y m e at the final stage h a d an a c t i v i t y of z . g p m o l e s / h / n d in the ribitol d e h y d r o g e n a s e assay a n d the. riUo~ ;.~,~merase a c t i v i t y wag o.7/~mole/h/ml o f enzyme. Properties of ~tw enzyme
W h e n the most purified e n z y m e was t e s t e d with v-ribose-5-P a n d D-ribulose-5-P ~.g the substrate, the results shown in Fig. 2 were obtained. These results i n d i c t t e t h a t the substrate o f t h e e n z y m e is ~-ribulose-5-P a n d not D-ribose-~-P. To confirm these observations, enzymic o x i d a t i o n of ribitol-5-P was carried out on a larger scale and the p r o d u c t identified b y a coiorimetric test a n d p a p e r chromatography. Tile reaction m i x t u r e contained 50 pmoles of Tris-HC1, z prnole of F~DTA, 5 mmoh~g of a m m o n i u m acetate, 24/~mo1~ o f ribitol-5-P, 2.5/~moles of T P N , zoo ~moles o f sodium pyruvate0 zo m g of crystalline lactic d e h y d r o g e n a s e a-Jd 75 u n i t s of e n z y m e in a volume of 8 ml (pH 7.8). After z h at 37 °. t h e solution was deproteinized b y the a d d i t i o n of z.5 ml of 4 o % triehloroacetic acid to give a p H o f 2.0. The precipitate was r e m o v e d h y c e n t r i f u g a t i o n at 3 °, a n d t h e s u p e r n a t a n t fluid neutralized vith Ba(OH)s t.o p H 6,5. After z h a t o ~, t h e pre=ipitate was r e m o v e d b y ccntrifugation a n d 4 vols. of e t h a n o l were added, After 4 h t h e precipitate was collected b y centrifugation, waslled with 8 o 0 e t h a n o l a n d ¢h'ied i n vacuo. The precipitate was dissolved in 0.03 M glycylglycine-o.oo3 M MgCIa (pH 7-5) a n d digested w i t h z m g o f E . coli phc~phatase for 3o rain. The solution was d~proteimzed w i t h perchloric acid a n d neutralized w i t h K O H , After r e m o v a l o f insoluble KCI04 b y centrifugation, t h e ~olution was deionized b y passage t h r o u g h an A m b e r lite MB-3 ¢ 91umn. The deionized solution co'~tained o . ~ 0 / , ~ o l e o f ketopentose per mlv a n d o.x45/amole/ml of aldopentosea. "l%e ketopentose color reached its m a x i m u m in Io mJn, the same as s n a u t h e n t i c ribulose s t a n d a r d ~s-~4. In the orcinol test t h e readings a t 540 m p a n d 670 m/~ were consis~'ent w i t h a m i x t u r e of ribulose a n d ribose in t h e above proportions ~. P a p e r c h r o m a t o g r a p h y o f this solution, using p h e n o l - w a t e r ( 4 : z ) as the solvent, indicated t w o c o m p o u n d s with t h e same Rw as a n t h e n t i c ribose a n d ribulose when s p r a y e d with aniline phosphateOL W h e n s p r a y e d with t h e orcinol reagent only a single ycUow spot w i t h t h e m o b i l i t y o f ribulose was seen which t u r n e d r e d on over-
Biockfm, B~o~Ays..4eta, 67 0963) 5z.~-~,3o
RIBITOL-5-P D E I I Y D R O G E N A S E
529
s p r a y i n g with aniline phos phat e. T h i s reaction is typical of ribulose b u t n~>t o f xylulos e " , re. Th es e resuRs show t h a t b o t h ribose-5-P a n d ribulose-5-P were o b t a i n e d b y t h e e n z y m i c o x i d a t i o n ofrib:A,,l 5 P. Uut t he ratio of ribulose-5-P t o ribose-5-P is x.3 : I a n d t h e r e f o r e r ib u lo s e-5- P is t h e p r i m a r y p r o d u c t of t he e n z y m e reaction, since at equilib r i u m in t h e r i b o ~ i s o m e r a ~ r eact i on this ratio would be 0.32 at 37 ° (see ref. Io). T o test w h e t h e r t h e red~tc*.i~m o f rihuio.~e-5-P by T P N H was stereospecific, r i b u l o s e - 5 - P was r e d u c e d w i t h T P N H a n d t h e r i b i t o l - 5 - P Ibrmed was isolated as t h e b a r i u m salt. T h e isolated m a t e r i a l was t r e a t e d with E. coli phosphata_~e and e h r o m a t o g r a p h e d using p y r i d i n e - e t h y l a c e t a t e - w a t e r (2: 7: : ) as t h e s o l v e n t ~. T h e enzymic_ally f o r m e d m a t e r i a l gave a single spot with t h e sam e R v as a u t h e n t i c ribitol, b u t di_stinct f r om arabitol, d b o s e a n d ribulose. Since arabito] i.-: t h e 2-epimer o f ribitol, these r ~ u i t s show t h a t t he e n z y m i c r e d u c t i u n of r i b u l o s c - 5 - P yields onl y t h e ribitol co n f ig ur a t i on a t C-2 of t he sngar alcohol. T h e e n z y m e will react w i t h D P N H or D P N instead ,,f T P N H o: T P N at a p p r o x . the s a m e rate. T P N H w a s used as a substrate, ~dnce tl,e apparent oxidase acti,.'ity in c r u d e e x t r a c t s is m u c h ie~s for T P N H t h a n for D P N H . A ft er t h e a m m o n i u m ~ulfate f r a c t i o n a t i u n t he en-gymc is free. o f T P N H oxid,~e a c t i v i t y . T h e e n z y m e will ~o • t r e a c t w i t h g l u c ~ e - 6 - P , fructose-6-1', ribose, ribulo~:, n b i t o | or ar~_bitol. Tb_e K m for r i b u l o ~ - 5 - P is ~.5. IO-t M (Fig. z). "/he Km fur T P N H is a p p r o x . 9" zo-S M. T h e e n z y m e is n ot s t i m u l a t e d b y the a d d i t i o n of Mg ~* and has a p H o p t i m u m o f 7.5. 0.2)5 /
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F i g . z. S u b s t r ~ t e s p e c i f i c i t y o f r l b i ~ o l - 5 - F d e h y d r o ~ e n a ~ o . T h e s t a u d a r d r ~ I c t i o n m i x t u r 0 w a s used with the following sul~tc a d d i t i o n s . C u r v e s A &nd C: o . t p m o l e o f r i b u l o ~ - S - P , t h o s o l u t i o n c o n t a i n e d o . 0 4 5 ~ m o l e o f r l b o s c - 5 - P . C u r v ~ B a n d D : o . x / , m o l e o f riboso-5-P, the s o l u t i o n c ~ n t a l r t e d o , o ~ 6 / ~ m o l e o f r / b u l o s e - 3 - P . All t u b ~ c o n t u s e d r/bitol-$+P dehytirogena~ p r e c i p i t a t e d a t p H 5- t. C u r v e g C a n d D c o n t a i n e d iu a d e P t / o n r i b o s e - $ - P isom~ra~ic.
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I~$ (S-,RlbulolooS-P M,~IO~ ) Fig. 3- A c t i v i t y of ribitol-5-P d e h y d r o g o n u e as tt f u n c t i o n of ribdlose-5-P concentratiort. S t a n d a r d assay s y s t e m ~ t t h s u b s tr a te concantr&tion as indicated. V -- ~ absorbamcy units a t 34 ° rata per rain. S ~ c o n c e n t r a t i o n o f r l b u t o s e - 5 - P {M x ~o'),
DISCUSSION
A large n u m b e r of p~lyol d e h y d r o g e n e ~ s h a v e been described from various n a t u r a l sources ~s, b u t with one exception* all the pentitol d e h y d r o g e n a - ~ a p p e a r to act on the free sugar.
In considering the biosynthesis o f ribitol-5-P in Line.bacillus pian~avum, the t w o obvious pc~sibilities were the reduction o f free ribose or ribulose to ribitol, followed by phosphorylation or a l t e r n a t i v e l y reduction of the p h o s p h o r y l a t e d pentose whic~ is t};e r e a c t i o n t h a t ha.~ b e e n d e s c r i b e d . W e h a v e b e e n u n a b l e t o d e t e c t a r i b i t o l d e h y d r o g e n a s e in e x t r a c t s o f t h i s o r g a n i s m . The procedure described for the preparation of ribitot-5-P dehydrogenase has
been reproducible, but different m e t h o d s o f preparing t h e cell-free e x t r a c t , such as the use of a sonic oscillator instead of a Nossal shaker, h a v e given e x t r a c t s w i t h much lower a c t i v i t y and sometimes completely inactive e x t r a c t s were obtained. ACKNO~,VLEDGItMENT This work was supported
by a grant (G-aoo2?) from the National Science Foundation.
REFEP, ENCES 1 j . BADDILEY AND A. P. ,Ai^TttI^S, J . Chem. So¢., (z954) 2723. s j . j . AaMSXaON~, J. H^DDlt.t~y, J. G+ BUCt4AI~'AN, A. L. D^WtSON. M. V. KV.Lt:M~:N AND F. C. N~UHAOS, Nature, ]84 (t959) z47. • i). I ( 1 ) . SHAW, Biochem. J., 82 (x962) z97, * }J. FAULKNER, Rio~hcm. j . , 64 (1956) 436. * B. L. H o a e c g ~ . a , in S. P. CocowxcK xuO N. I), KAPLAN, Methods in En,ymology, Vol 3, A c a d e m i c Press, N e w York. t957, P. zgo. * A. H. Bmowl¢, Arch. B*ochem. Biopkys., ~! (tg52) 369. f Z. DISCHI AICD E. BOItENFREUND, J. Biol. Ck4em.. 19~t ft951) 583.
* Z. D]scHR AICD E. BOReNFR~Ut~D, Biockim. Biophys. Aota, a3 (x957) 639'* J. BADDILBY, J. G. BUctlANe~s, B. CAtlut t, lCD A. P. MAT:I.IAB, J . Ck0rtt. so l .. (i95(~) 4583. ]* B. AxtttJtOD AND R. JANO, J. Biol. C[~tm., 209 (~9;54) 847. 7t O. ~A'ARBUFtO^Z~t) W. CnalsTt^,q. Biochem. Z,, 3xo (1941) 384. lu S. COHES, J. Biol. C/tom., ~tol (t953} 7 I. 7. G. A~ttwxLl. ,~¢t) J. HICKMANtq, J . 4m. Ck4tm. So0., 76 [t954) 5889. 7, p. K. S~rumpr AND B. L. HoItttCK,,a, J. B ~ . Ck*m., ~ t 8 {1956) 753. 17 B. L. H o a ~ t c g ~ t . P. 2. St4vtucxoTm AND j. E. S z a o m ~ . n , J. B~ot. ¢~m.. t93 ('95~) 383. ~* F. DTcxz3ca ^I¢D D , H. W I u . x t m a o s , Bio~Aam. J , , 64 (1956) 567. x~ it. K~.~VSTn^ND AND A. NORn,~.. Acta Ck~tm. S c ~ t , . 4 (x95o) x32o. I* O. TOUI~TgR A~qDD. R. D. Sit^w, PAytiol. R#v,, 4Z (196a) %8].
Oiockim. Biophy~, Acta, 67 (zc~b3) 5a5-53o
~2~
BBA xzz93 RIBITOL-5-FHOSPHATE DEHYDROGENASE F R O M L A C T O B A C I L L U.q P L A N T A R U ~ I LUIS GLASER
Da'partr~=l of Biological Ckem~try. Wash~n~'~. Uni~er_~,ly School of ,~qr~i¢i,w. .S'ai~t Louis. Me+ (U.S.A./ ( R e c e i v e d J u l y tGth. I S Z j
S U M M A I~a(
T h e partial purification o f a r i b i t o L s - p h o s p h a t e d e h y d r o g e n a t e from Lactob.~cill,,s plant.arm (ATCC 8oi4) is described. The e n z y m e has linen shown to catalyze the reaction TPN ~ TPNH r~--~bitol 3-phcmphe~t~ + or ,# D-ribu[o~e 5-|>hosph~,t,: + or + ]{+ DPN ~
DPNH
INTRODUCTION
I n 1954 BADDILEY ^~D MATHIASx described the isolation of COP-ribitol and later the presence o f ribitol teichoic acids (a family of sub~titutexi polyribito! phosphates) in a v a r i e t y of gram-positive b a c t e r i # . T h e enzymic svnthesis of CDP-ribitol from D-ribitol-5-P a n d C T P has been described b y SHAWa. No e n z y m ~ which specifically catalyze t h e synthesis of D-ribitol-5-P have been isolated although an a p p a r e n t l y tmspecific aldose reductase from silkworm blood will catalyze the reduction of or i b o s e - 5 - P to D-ribitol-5-P (see ref. 4)- W e wish to report the partial purification of a n e n z y m e which c a t a l ) ~ s the formation oi ~ b i t o l - 5 - P from D-ribulose.3-P, using either D P N H or T P N H as a h y d r o g e n donor. EXPERIMENTAL
Mataria2s Laa0bact'Uus #Lantarum (ATCC 8o14) was g r o w n in a m e d i u m oQntalning per 1: 4 g o f Difco y e a s t extract, xo g o f Difco nutrient broth, ro g of r ~ l i u m acetate. o.a g o f MgSO,. 7 H a O , o.ox g o f NaCI, o.o! g of Fea(SOi)s. 9 HtO, o.oz g o f MnSOa" 7 H t O a n d ao g o f glucose. An m o c u i u m from a ~iab w ~ g~o'~.~ in Too nfl of this medimm~for xa h, a n d 6 ml -,f this c u l t u r e were used to inoculate 3 ! o f m e d i u m in a 6-! E r l e n m z y e r flask a n d t h e culture allowed to grow for xz h at 37 °. The ceils from x8 1 o f c u l t u r e m e d i u m were bin-vested b y centrifugaGnn a n d w a s h e d tw~:ce with 4oo nfl of o.o5 M T~'~-HCl~o.oot M E D T A ( p i t 8.o). The ceEs were fmaUy suspended in a mininml v o l u m e o f o.o5 M Tris~HCl-o.ox M MgCtt-o.ooz M E D T A ( p H 6-0) ~ d Biockim. Bio#t~5. Aaa. 67 ~1963) 5 ~ 5 - 5 3 o
5z6
t,. OLAsER
lyophilizcd. The lyoph'"-flized powder could be stored for at least 3 weeks at ~ 2 o ° without loss of activity. ~ . R i b u l o ~ - 5 - P was prepared b y the m e t h o d of Hom~cKat~'. The 6-phosphogluconic acid d e h y d r o g e n a s e s used contained ritxJ~-5-P isomcrase and the final product, even after column c h r o m a t o g r a p h y , was c o n t a m i n a t e d with some ribose-5-P. Pentose was d e t e r m i n e d b y the orcinol m e t h o d e, with a 4o-min heating period in a volume of r.5 ml. Ribulose was determined b y the cysteine carbazole reaction ~ using a u t h e n t i c r i b d o s e as a standard. R i b u l o s e - 5 - P was d e t e r m i n e d b y the same m e t h o d after r e m o v a l of the p h o s p h a t e with chromatographically purified E-chcrichia coil phosphatase (Wowthington Biochemical Corporation), thus permitting the use of free ribulose as a s t a n d a r d ~n ille colorimetric assay. Aldopenta.~es were d e t e r m i n e d b y the phloroglucittol m e t h o d n. t~-Ribnh~e-o-nitrophenylhydrazone was obtainc~l from California Corporation for Bi~ychc-m~cal Re.~arch. Ribose-5-F from S c h w a r t z Laboratories, D P N H a n d T P N H were obtained from the Sigma Chemical Company, R i b i t o l - f - P was p r e p a r e d b y the methtxl of BADDILEY tit a / , 9. R i b o s e - 5 - P isomerase wa_~ d e t e r m i n e d b y incubating enzyme with ribose-5-P (see ref. to) under the conditions of the ribitol-5-P d e h y d r o g e n a . ~ assay. At t h e end of ineuV:ation, the sugar p h o s p h a t e s were h y d r o l y z e d with E. coli p h o s p h a t a ~ and the ribulose formed determinedL Protein was d e t e r m i n e d b y the m e t h o d o f WAI~BUaC; :_XD CXmSTl^N n. P r o t a m i n e sulfate was obtained from Si~,ma Chemical Company. Fresh solutions of p r o t a m i n e sulfate were prepared for the e n z y m e fractior~ti_~n and neutralized to p H 7.o with K O H .
As.say of ribitol-5-P dehydrogenase The reaction ntixture contained: t o # m o l e s of T n s - H C I , o.2 l,mole o f E D T A , 0.3/1mole of ribulo.se-5-P , o.x/~mole of T P N H and 500/~moles of 0anmonium a c e t a t e in a final v o l u m e of x ml {pH 7.8). Control reaction m i x t u r e s c o n t a i n e d 811 t h e c o m p o n e n t s minus ribulose-5-P. A b ~ r b a n c y m e a s u r e m e n t s were t~keP at 340 m/~ ever 3' m i n u t e in a B e c k m a n s p e c t r o p h o t o m e t e r t h e r m o s t a t e d at z5 °. I n all the e n z w n e fractions except the final p H - 5 precipitate (see below) ribose-5-P could s u b s t i t u t e for ribulose-5-P w i t h o u t change of activity. The e n z y m e could also be assayed b y measuring T P N H formation from ribitol5 - P and T P N . The reaction m i x t u r e c o n t a i n e d x o / a n o l e s of Tris--HCl, o.~ p~nole o f E D T A , z #moles of ribitol-5-P, 5 / l m o l e s of T P N and 5 o o ~ m o l e s of a m m o n i u m a c e t a t e in a final volume of I ml (pH 7.8). W i t h the crude extract, values o b t a i n e d b y this &ssay are low due to the presence of T P N H oxidase. I unit is defined as t h e a m o u n t of e n z y m e required t o catalyze a change of o.ox a b s o r b a n c y unit/rain. T h e d e p e n d e n c e of the a c t i v i t y on e n z y m e concentration is shown in Fig. z. RESU LT~
PHrifu:ation of ribitol-5-P dehydrogcnas¢" x g of d r y cells was suspended in 8 ml of o.o5 M Tris-HC1, o.oz M MgC11. o.oox M • All s t e p s i n t h e e n z y m e p u r i f i c a t i o n w e r e c a r r i e d o u t a t 3 °. A• p r e c i p i t a t o r s w e r e c o l l e c t e d b y c e n t r t f u g a t i o n a t z2 c o o × g f o r t o r a i n . A m m o n i u m s u l f a t e w a s ~atur~Lt~t a t 3 ° a n d n e u ~ z e d w i t h . N ' H . O H s o t h a t a I : 5 d i l u t i o n h a d a p H c~f 7.5-
Biockim. Biophys. A~:ta, (17 0903) 5~5-~$o
R! BITOi.-~-~"
DEHYDROGENASE
527
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Fig. t. t)cpcndencc of ribitol-5-P dehydrogena~v activity on enzyme c~,,centration. Stand~r0 assay conditions; tit.: enzyme was ammonium :~ulphatc ftatt:tion. E D T A ( p H 8.o), s h a k e n w i t h 8 g o f glass h e a d s in a Nossal s h a k e r for t h r e e 3n-see p e r i o d s b e i n g c o o l e d in b e t w e e n e a c h 3o-.~ec p e r i o d in a n ice b a t h . T o p r e v e n t fc~'aming. a d r o p o f c a p r y l a l c o h o l w a s a d d e d . T h e gla_~s h e a d s w e r e allowed to s e t t l e a n d t h e s a i p e r n a t a n t fluid c e n t r i f u g e d . T h e b e a d s a n d p r e c i p i t a t e w e r e wa_~hed t w i c e w i t h 5 m l o f t h e s a m e buffer. T h e p o o l e d s u p e r n a t a n t fluid was t h e n f r a c t i o n a t e d w i t h p r o t a m m e sulfate. In a t y p i c a l e x p e r i m e n t as show.~ in T a b l e I. I8. 5 ml o f 2 % p r o t a r n i n e s u l f a t e was a d d e d t o t h e e n z y m e ar.d a f t e r in rag,, t h e p r e c i p i t a t e wa* c o l l e c t e d b y ce,Lt~ifugali,,, T AI3LE l .
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a n d d i s c a r d e d . T o t h e s u p e r n a t a n t fluid a n a d d i t i o n a l i 6 m l o f 2 % p r o t a m i r ~ s u l f a t e w e r e ad.:~-,d a n d a f t e r I n rain t h e p r e c i p i t a t e w,~s c o l l e c t e d b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a . l t fluid d i s c a r d e d . T h e p r e c i p i t a t e f r o m t h e s e c o n d p r o t a m i n e a d d i t i o n was w a s h e d w i t h 2o ml o.~ M a m m o n i u m a c e t a t e ( p H 7.8) a n d t h e n e x t r a c t e d for 0. 5 h w i t h z5 m | o f x M a m m o n i m . , a c e t a t e ( p H 7.8). T h e i n s o l u b l e r e s k l u e was r e m o v e d b y c e n t r i f u g a t i o n a n d d i s c a r d e d . T o t h e s u p e r n a t a n t fluid, c o n t a i n i n g t h e e n z y m e , w a s a d d e d a n e q u a l v o l u m e o f s a t d . a m m o n i u m sulfate, a n d a f t e r iS rain t h e precib,~:.~- w a s colle."t 1 b y c e n t r i f u g a t i o n a n d d i s s o l v e d in x M a m m o n i u m a c e t a t e ( p H 7.8j. T h e , ' - t z y m e w a s t h e n d i a l y z e d a g a i n s t o . I 5 M a c e t a t e b u f f e r ( p H 5.x) w i t h
Bio~Mm. Iiiop#tys. AcM. 67 (x963) 525-53 °
5Z~
L, GLASER
stirring for 4 h. FinaLly the precipitate was collected b y centrifugation a n d dis.~olved in ~ M a m m o n i u m acetate (pH 7,8). The e n z y m e can be k e p t frozen at all stages in t h e purification for at least a week w i t h o u t loss o f activity. The s u m m a r y o f a typical p r e p a r a t i o n is s h o w n in Table I. I t has been folma necessary to pilot the p r o t a m i n e step w i t h ever3- e n z y m e preparation, siuce ~.b.z quanL~ty o f p r o t a m i n e which will precipitate m~x~t o f t h e nucleic acid b u t not the e n z y m e , varies from p r e p a r a t i o n to preparation. After the p r o t a m i n e step the e n z y m e becomes u n s t a b l e at low ionic strength° a n d dialysis of this fraction against o,z M p h o s p h a t e (pH 7.5) or o.z M Tris (pH 7-5) for 4 h leads to complete loss of acti-rity. Precipitation at p H 5.1 causes a relatively large loss of a c t i v i t y b u t serves to r e m o v e m o s t of the vibose-3-P isomerase. Reprecipitation o f the e n z y m e a t p H 5.z does not remove the last traces o f isomerase. In t h e p r e p a r a t i o n shown in Table l, the e n z y m e at the final stage h a d an a c t i v i t y of z . g p m o l e s / h / n d in the ribitol d e h y d r o g e n a s e assay a n d the. riUo~ ;.~,~merase a c t i v i t y wag o.7/~mole/h/ml o f enzyme. Properties of ~tw enzyme
W h e n the most purified e n z y m e was t e s t e d with v-ribose-5-P a n d D-ribulose-5-P ~.g the substrate, the results shown in Fig. 2 were obtained. These results i n d i c t t e t h a t the substrate o f t h e e n z y m e is ~-ribulose-5-P a n d not D-ribose-~-P. To confirm these observations, enzymic o x i d a t i o n of ribitol-5-P was carried out on a larger scale and the p r o d u c t identified b y a coiorimetric test a n d p a p e r chromatography. Tile reaction m i x t u r e contained 50 pmoles of Tris-HC1, z prnole of F~DTA, 5 mmoh~g of a m m o n i u m acetate, 24/~mo1~ o f ribitol-5-P, 2.5/~moles of T P N , zoo ~moles o f sodium pyruvate0 zo m g of crystalline lactic d e h y d r o g e n a s e a-Jd 75 u n i t s of e n z y m e in a volume of 8 ml (pH 7.8). After z h at 37 °. t h e solution was deproteinized b y the a d d i t i o n of z.5 ml of 4 o % triehloroacetic acid to give a p H o f 2.0. The precipitate was r e m o v e d h y c e n t r i f u g a t i o n at 3 °, a n d t h e s u p e r n a t a n t fluid neutralized vith Ba(OH)s t.o p H 6,5. After z h a t o ~, t h e pre=ipitate was r e m o v e d b y ccntrifugation a n d 4 vols. of e t h a n o l were added, After 4 h t h e precipitate was collected b y centrifugation, waslled with 8 o 0 e t h a n o l a n d ¢h'ied i n vacuo. The precipitate was dissolved in 0.03 M glycylglycine-o.oo3 M MgCIa (pH 7-5) a n d digested w i t h z m g o f E . coli phc~phatase for 3o rain. The solution was d~proteimzed w i t h perchloric acid a n d neutralized w i t h K O H , After r e m o v a l o f insoluble KCI04 b y centrifugation, t h e ~olution was deionized b y passage t h r o u g h an A m b e r lite MB-3 ¢ 91umn. The deionized solution co'~tained o . ~ 0 / , ~ o l e o f ketopentose per mlv a n d o.x45/amole/ml of aldopentosea. "l%e ketopentose color reached its m a x i m u m in Io mJn, the same as s n a u t h e n t i c ribulose s t a n d a r d ~s-~4. In the orcinol test t h e readings a t 540 m p a n d 670 m/~ were consis~'ent w i t h a m i x t u r e of ribulose a n d ribose in t h e above proportions ~. P a p e r c h r o m a t o g r a p h y o f this solution, using p h e n o l - w a t e r ( 4 : z ) as the solvent, indicated t w o c o m p o u n d s with t h e same Rw as a n t h e n t i c ribose a n d ribulose when s p r a y e d with aniline phosphateOL W h e n s p r a y e d with t h e orcinol reagent only a single ycUow spot w i t h t h e m o b i l i t y o f ribulose was seen which t u r n e d r e d on over-
Biockfm, B~o~Ays..4eta, 67 0963) 5z.~-~,3o
RIBITOL-5-P D E I I Y D R O G E N A S E
529
s p r a y i n g with aniline phos phat e. T h i s reaction is typical of ribulose b u t n~>t o f xylulos e " , re. Th es e resuRs show t h a t b o t h ribose-5-P a n d ribulose-5-P were o b t a i n e d b y t h e e n z y m i c o x i d a t i o n ofrib:A,,l 5 P. Uut t he ratio of ribulose-5-P t o ribose-5-P is x.3 : I a n d t h e r e f o r e r ib u lo s e-5- P is t h e p r i m a r y p r o d u c t of t he e n z y m e reaction, since at equilib r i u m in t h e r i b o ~ i s o m e r a ~ r eact i on this ratio would be 0.32 at 37 ° (see ref. Io). T o test w h e t h e r t h e red~tc*.i~m o f rihuio.~e-5-P by T P N H was stereospecific, r i b u l o s e - 5 - P was r e d u c e d w i t h T P N H a n d t h e r i b i t o l - 5 - P Ibrmed was isolated as t h e b a r i u m salt. T h e isolated m a t e r i a l was t r e a t e d with E. coli phosphata_~e and e h r o m a t o g r a p h e d using p y r i d i n e - e t h y l a c e t a t e - w a t e r (2: 7: : ) as t h e s o l v e n t ~. T h e enzymic_ally f o r m e d m a t e r i a l gave a single spot with t h e sam e R v as a u t h e n t i c ribitol, b u t di_stinct f r om arabitol, d b o s e a n d ribulose. Since arabito] i.-: t h e 2-epimer o f ribitol, these r ~ u i t s show t h a t t he e n z y m i c r e d u c t i u n of r i b u l o s c - 5 - P yields onl y t h e ribitol co n f ig ur a t i on a t C-2 of t he sngar alcohol. T h e e n z y m e will react w i t h D P N H or D P N instead ,,f T P N H o: T P N at a p p r o x . the s a m e rate. T P N H w a s used as a substrate, ~dnce tl,e apparent oxidase acti,.'ity in c r u d e e x t r a c t s is m u c h ie~s for T P N H t h a n for D P N H . A ft er t h e a m m o n i u m ~ulfate f r a c t i o n a t i u n t he en-gymc is free. o f T P N H oxid,~e a c t i v i t y . T h e e n z y m e will ~o • t r e a c t w i t h g l u c ~ e - 6 - P , fructose-6-1', ribose, ribulo~:, n b i t o | or ar~_bitol. Tb_e K m for r i b u l o ~ - 5 - P is ~.5. IO-t M (Fig. z). "/he Km fur T P N H is a p p r o x . 9" zo-S M. T h e e n z y m e is n ot s t i m u l a t e d b y the a d d i t i o n of Mg ~* and has a p H o p t i m u m o f 7.5. 0.2)5 /
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F i g . z. S u b s t r ~ t e s p e c i f i c i t y o f r l b i ~ o l - 5 - F d e h y d r o ~ e n a ~ o . T h e s t a u d a r d r ~ I c t i o n m i x t u r 0 w a s used with the following sul~tc a d d i t i o n s . C u r v e s A &nd C: o . t p m o l e o f r i b u l o ~ - S - P , t h o s o l u t i o n c o n t a i n e d o . 0 4 5 ~ m o l e o f r l b o s c - 5 - P . C u r v ~ B a n d D : o . x / , m o l e o f riboso-5-P, the s o l u t i o n c ~ n t a l r t e d o , o ~ 6 / ~ m o l e o f r / b u l o s e - 3 - P . All t u b ~ c o n t u s e d r/bitol-$+P dehytirogena~ p r e c i p i t a t e d a t p H 5- t. C u r v e g C a n d D c o n t a i n e d iu a d e P t / o n r i b o s e - $ - P isom~ra~ic.
B~o~Mm. Biop/r~s. ,4aa, 6 7 ( , 9 6 3 ) 5 2 5 - 5 3 0
530
L. GLASER
'2I0 ......................... 0.04 O.OB
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I~$ (S-,RlbulolooS-P M,~IO~ ) Fig. 3- A c t i v i t y of ribitol-5-P d e h y d r o g o n u e as tt f u n c t i o n of ribdlose-5-P concentratiort. S t a n d a r d assay s y s t e m ~ t t h s u b s tr a te concantr&tion as indicated. V -- ~ absorbamcy units a t 34 ° rata per rain. S ~ c o n c e n t r a t i o n o f r l b u t o s e - 5 - P {M x ~o'),
DISCUSSION
A large n u m b e r of p~lyol d e h y d r o g e n e ~ s h a v e been described from various n a t u r a l sources ~s, b u t with one exception* all the pentitol d e h y d r o g e n a - ~ a p p e a r to act on the free sugar.
In considering the biosynthesis o f ribitol-5-P in Line.bacillus pian~avum, the t w o obvious pc~sibilities were the reduction o f free ribose or ribulose to ribitol, followed by phosphorylation or a l t e r n a t i v e l y reduction of the p h o s p h o r y l a t e d pentose whic~ is t};e r e a c t i o n t h a t ha.~ b e e n d e s c r i b e d . W e h a v e b e e n u n a b l e t o d e t e c t a r i b i t o l d e h y d r o g e n a s e in e x t r a c t s o f t h i s o r g a n i s m . The procedure described for the preparation of ribitot-5-P dehydrogenase has
been reproducible, but different m e t h o d s o f preparing t h e cell-free e x t r a c t , such as the use of a sonic oscillator instead of a Nossal shaker, h a v e given e x t r a c t s w i t h much lower a c t i v i t y and sometimes completely inactive e x t r a c t s were obtained. ACKNO~,VLEDGItMENT This work was supported
by a grant (G-aoo2?) from the National Science Foundation.
REFEP, ENCES 1 j . BADDILEY AND A. P. ,Ai^TttI^S, J . Chem. So¢., (z954) 2723. s j . j . AaMSXaON~, J. H^DDlt.t~y, J. G+ BUCt4AI~'AN, A. L. D^WtSON. M. V. KV.Lt:M~:N AND F. C. N~UHAOS, Nature, ]84 (t959) z47. • i). I ( 1 ) . SHAW, Biochem. J., 82 (x962) z97, * }J. FAULKNER, Rio~hcm. j . , 64 (1956) 436. * B. L. H o a e c g ~ . a , in S. P. CocowxcK xuO N. I), KAPLAN, Methods in En,ymology, Vol 3, A c a d e m i c Press, N e w York. t957, P. zgo. * A. H. Bmowl¢, Arch. B*ochem. Biopkys., ~! (tg52) 369. f Z. DISCHI AICD E. BOItENFREUND, J. Biol. Ck4em.. 19~t ft951) 583.
* Z. D]scHR AICD E. BOReNFR~Ut~D, Biockim. Biophys. Aota, a3 (x957) 639'* J. BADDILBY, J. G. BUctlANe~s, B. CAtlut t, lCD A. P. MAT:I.IAB, J . Ck0rtt. so l .. (i95(~) 4583. ]* B. AxtttJtOD AND R. JANO, J. Biol. C[~tm., 209 (~9;54) 847. 7t O. ~A'ARBUFtO^Z~t) W. CnalsTt^,q. Biochem. Z,, 3xo (1941) 384. lu S. COHES, J. Biol. C/tom., ~tol (t953} 7 I. 7. G. A~ttwxLl. ,~¢t) J. HICKMANtq, J . 4m. Ck4tm. So0., 76 [t954) 5889. 7, p. K. S~rumpr AND B. L. HoItttCK,,a, J. B ~ . Ck*m., ~ t 8 {1956) 753. 17 B. L. H o a ~ t c g ~ t . P. 2. St4vtucxoTm AND j. E. S z a o m ~ . n , J. B~ot. ¢~m.. t93 ('95~) 383. ~* F. DTcxz3ca ^I¢D D , H. W I u . x t m a o s , Bio~Aam. J , , 64 (1956) 567. x~ it. K~.~VSTn^ND AND A. NORn,~.. Acta Ck~tm. S c ~ t , . 4 (x95o) x32o. I* O. TOUI~TgR A~qDD. R. D. Sit^w, PAytiol. R#v,, 4Z (196a) %8].
Oiockim. Biophy~, Acta, 67 (zc~b3) 5a5-53o