Pyruvate decarboxylase in photosynthetic bacteria

304

BIOCHIMICA ET BI()PHYSIC.~ .\(T,\

PRELIMINARY

NOTES

9 BBA _1188

Pyruvate decarboxylase in photosynthetic bacteria I n t h e course ()f some investigations on p y r u v a t e a n d f o r m a t e m e t a b o l i s m b y an isolate of a purple non-sulfur p h o t o s y n t h e t i c b a c t e r i u m , Rhod@seudomonas pahtslrzs I s t r a i n Q, it was found t h a t p y r u v a t e was d e c a r b o x y l a t e d a n a e r o b i c a l l y w i t h o u t the formation of hydrogen. This was in c o n t r a s t to e x p e r i m e n t s with the purple sulfur b a c t e r i u m Chromatium which c a t a l y z e d a phosphoroclastic degradati(m of pyruvate2, a. F u r t h e r e x a m i n a t i o n of p y r u v a t e d e g r a d a t i o n b y cell-free e x t r a c t s of R. flalustris Q, have e s t a b l i s h e d t h a t the p r o d u c t s of the reaction are CO,,, acetaldehyde and acetylmethylcarbin()l (acetoin). R. flalustris Q thus appears t,) contain p y r u v a t e d e c a r b o x y l a s e (2-oxoacid earboxy-lyase, EC 4 . I . I . I ) , an enzyme which is not conlinonly found in bacteria, and which has not p r e v i o u s l y been r e p o r t e d in a p h o t o s y n t h e t i c b a c t e r i u m . This p r e l i m i n a r y report provides evidence for tile presence of a t h i a m i n e p y r o p h o s p h a t e (TPP) a c t i v a t e d p y r u v a t e d e c a r b o x v l a s e in e x t r a c t s ()f some species of purple non-sulfur b a c t e r i a (Athiorhodaceae). Fig. I shows the t i m e course for the reaction with at cell-free e x t r a c t ()f 1¢. pahtstris O. The organisnl was grown a n a e r o b i c a l l y in the light at 28 `> (m a basal m e d i u m 4 with sodium l a c t a t e as m a j o r source of organic carbon. ('ell-fret e x t r a c t s were p r e p a r e d b y sonic d i s r u p t i o n in 5o mM p o t a s s i u m p h o s p h a t e buffer (pH !3.e5. followed b y centrifugations for 3 ° rain at 34800 . ,q* and 6o rain at 9oooo .4'. The clear p i g n l e n t e d e x t r a c t s were used for assays. Protein concentrations d e t e r m i n e d from the a b s o r b a n e e at 260 and 280 roll (ref. 5) were in the range 5-IO lug per ml. F o r m a t i o n of a c e t a l d e h y d e has been confirmed in a larger scale e x p e r i m e n t in which the reaction m i x t u r e (2o ml c o n t a i n i n g IOO /,moles p y r u v a t e ) was a e r a t e d c o n t i n u o u s l y at 2 6 ' with a gentle s t r e a m (>f argon for 3 h, and the a c e t a l d e h y d e was t r a p p e d in 5 ° mM p o t a s s i u m p h o s p h a t e but~fer (pH 6.25) at i .... A c e t a l d e h y d e , unc o n t a m i n a t e d b y other r e a c t a n t s or products, was then e s t i m a t e d b y three pr()cedures' (I) as the d i n i t r o p h e n y l h y d r a z o n e v (e) b y reaction with piperazine and s()dium m t r o p r u s s l d e , , (3) b y reaction with f l - h y d r o x y d i t ) h e n y l and H2S()~ TM. (;.<)d agree m e n t between estimati(ms b y these three m e t h o d s was obtained. l>yruvate d e c a r b o x y l a s e of R. ySahtstris Q had an o p t i m u m p H ()f t).25 in 5o mM p o t a s s i u m phosphate, buffer, b u t lost a c t i v i t y irreversibly when dialyzed overnight against IO mM p o t a s s i u m phost)hate buffer (pH 6.25) at '2<>. If extra(:ts were d i a l y z e d at 2 ° u n d e r a n a e r o b i c c o n d i t i o n s (under argon) against IO mM buffer (pH ~>.25) c,))> t a i n i n g I mM cysteine, the loss in a c t i v i t y was restored b v the a d d i t i o n of t h i a m i n e p y r o p h o s p h a t e without the further a d d i t i o n of a d i v a l e n t metal ion. E x t r a c t s of b a k e r ' s y e a s t (Fleischman) p r e p a r e d under identical conditions required b o t h t h i a m i n e p y r o p h o s p h a t e and a d i v a l e n t m e t a l ion to restore full a c t i v i t y to d i a l y z e d extracts, a n d anaerobic c()nditions were not essential t() p r e v e n t irreversible loss ,,f a c t i v i t y ('Fable I). •

'

9

Abbreviation : TPP, thiamine pyrophosphate.

t3iochim. 13iophvs. A cla, 148 (1907) 3o4 3o0

P R E L I M I N A R Y NOTES 20

30~

I

PYRUVATE

co hi .--I

o ic

ACETALDEHYDE

::L

I

I

I

I

30

60

90

120

NL______-2 150

180

MINUTES

F i g . I. T i m e c o u r s e of p y r u v a t e d e c a r b o x y l a s e i n a n e x t r a c t of R. palustris Q. W a r b u r g f l a s k s c o n t a i n i n g i n a r e a c t i o n m i x t u r e of 3 m l (/,moles) : S o d i u m p y r u v a t e 20, p o t a s s i u m p h o s p h a t e b u f f e r ( p H 6.25) 14o , a n d e x t r a c t (12 m g p r o t e i n ) , w e r e i n c u b a t e d a n a e r o b i c a l l y u n d e r a r g o n a t 3 o°. T h e r e a c t i o n w a s s t o p p e d b y a d d i n g o . i m l 5 0 % ( w / v ) t r i c h l o r o a c e t i c a c i d f r o m a s e c o n d s i d e - b u l b . T h e f l a s k s w e r e c h i l l e d in ice a n d t h e r e a c t i o n p r o d u c t s e s t i m a t e d o n d e p r o t e i n i z e d s o l u t i o n s . P y r u v a t e a n d a c e t a l d e h y d e w e r e e s t i m a t e d b y m o d i f i c a t i o n s of t h e d i n i t r o p h e n y l h y d r a z o n e p r o c e d u r e s of L u 6 a n d of GREENBERG A N D L E S T E R 7, r e s p e c t i v e l y ; a c e t o i n w a s e s t i m a t e d b y t h e m e t h o d of WESTERFELD 8. (~)- - O , p y r u v a t e ; & - - & , CO 2 ; [] - - [], a c e t a l d e h y d e ; • - - • , acetoin. TABLE

I

STABILITY AND ACTIVITY OF PYRUVATE DECARBOXYLASE IN DIALYZED EXTRACTS OF YEAST AND

R. palustris Q Cell-free e x t r a c t s p r e p a r e d a s d e s c r i b e d i n t h e t e x t a n d p y r u v a t e d e c a r b o x y l a s e a c t i v i t y d e t e r m i n e d m a n o m e t r i c a l l y u s i n g t h e r e a c t i o n m i x t u r e i n d i c a t e d i n t h e l e g e n d t o Fig. I w i t h a d d i t i o n s a s shown below.

Extract treatment

Additions (#moles~3 ml)

Specific activity (Qeo2) Yeast

R. palustris (~

N o n e (fresh) 18 h s t o r e d a t 2 °

None None

7° 66

14 13

D i a l y z e d 18 h a t 2 ° a g a i n s t i o m M KzPO4, p H 6.25

None TPP, o.i MnC12, i . o T P P + MnCI~

19 3° 19 66

1. 5 1. 9 1.5 2. 5

D i a l y z e d 18 h a t 2 ° a g a i n s t IO m M K3PO4, p H 6.25, + i m M c y s t e i n e under argon

None TPP, o.i MnCI 2, I.O T P P + MnC12

19 31 20 66

2.0 12 2.2 12

Biochim. Biophys. Acta, 148 (1967) 3 o 4 - 3 o 6

306

PRELIMINAI¢Y NOTES

I n a p r e l i m i n a r y s u r v e y of s o m e A t h i o r h o d a c e a e g r o w n u n d e r t h e s a m e c o n d i p y r u v a t e d e c a r b o x y l a s e w a s also f o u n d in Rhodopseudomonas gelatinosa, Rhod@seudomonas capsulata, Rhodo@irillum m~.lischianum, a n d in o n e of V=~x NIEL's s t r a i n s of R. palustris; it was n o t f o u n d in Rhodopseudomonas spheroides, Rhodo@irillum rubrum or in Rhodomicrobium vannielii. I t is s i g n i f i c a n t t h a t R. r~tbrum, in M i l c h we h a v e f a i l e d to d e t e c t a p y r u v a t e d e c a r b o x y l a s e , h a s b e e n s h o w n to d e c o m p o s e p y r u v a t e t o CO 2 a n d w d a t i l e f a t t y a c i d s ( m a i n l y a c e t a t e a n d p r o p i o n a t e ) l l . E a r l i e r s t u d i e s o n t h e m e t a b o l i s m of f i - t q ~ l a c t a t e in t h e l i g h t b y a s t r a i n of R. pahtstris s h o w i n g t h e f o r m a t i o n ()f la(;() 2 via p y r u v a t e , c a n n o w b e i n t e r p r e t e d t o p r o c e e d via t h e a c t i o n of p y r u v a t e d e c a r b o x v l asO". P y r u v a t e d e c a r b o x y l a s e h a s a v e r y l i m i t e d d i s t r i b u t i o n in b a c t e r i a a n d a p a r t f r o m t h e p h o t o s y n t h e t i c b a c t e r i a r e p o r t e d h e r e , it h a s o t h e r w i s e b e e n f o u n d o n l y in tions,

Zvmosarcina ventricuh, Zvmomonas mobilis, Zvmom.onas anaerobia, Erwinia amvlovora 'a a n d t h e a c e t i c a c i d b a c t e r i a ~4,'s. T h e p h o t o s y n t h e t i c b a c t e r i a e v i d e n t l y fall i n t o t w o g r o u p s o n t h e b a s i s of p y r u v a t e d e e a r b o x v l a s e c o n t e n t . T h e p o s s i b l e t a x o n o m i c a n d p h y s i o l o g i c a l s i g n i f i c a n c e of its d i s t r i b u t i o n in p h o t o s y n t h e t i c m i c r o o r g a n i s m s r e m a i n s to be d e t e r m i n e d . O c c u r r e n c e of p y r u v a t e d e c a r b o x v l a s e in p h o t o s y n t h e t i c b a c t e r i a is p a r t i c u l a r l y u n u s u a l b e c a u s e w i t h t h e e x c e p t i o n of t h e a c e t i c a c i d b a c t e r i a , t h e p r e s e n c e (~f p y r u v a t e d e c a r b o x v l a s e h a s b e e n r e s t r i c t e d t o m i e r o o r g a n i s n l s w i t h a f e r m e n t a t i v e m e t a b o l i s m p r o d u c i n g e t h a n o l as a f e r m e n t a t i o n product. T h i s w o r k w a s s u p p o r t e d b y G r a n t A I o~)447 f r o m t h e U.S. I ' u b l i c H e a l t h S e r v i c e . W e a r e i n d e b t e d t o XZrs. A s h " HARBIS()X for t e c h n i c a l a s s i s t a n c e .

l)@artment of Microbiology, The University of Texas, Austin, Texas (U.S.A.I

S. M. H u s s M N QADRt I). S. HOARF~

I S. 3I. ]-[. (~)ADRI AND I). S. HOARE, Bacteriol. Proc., (1967) , 2 ~ . 1{. I{ENNETT, X. ]{IGOPOULOS AND |{. ('. FUI.LI~;R, ])t'oC..\'tlff. dead.

2 3 4 5 ~, -

Nci. Ir.N., 52 ([()(q) 71~;. l{. BENNETT .\ND l~.. ('. I:ULLER, lqiochim. Biopk3's. t?es. ('om,zu~., lO (~90i) 3oo. C. B. VAN NIEL, Bac[cJiol. l~ev., S (1944) I. (). \VAR~:RG AND \V. CHRISTIAN, l~iochem. Z., 31o (I942) 3S4 . G. D. l.u, tHockem, ft., 33 (1939) 24t). L . . \ . (;REENBJ~;RG AND [). I.ESTER, J. ]~iO[. Chem., 154 (t94,1) I77.

~q \ g . \ g . \ V E S T E R F E L D , jr. ~ i 0 l . Ckem., r(,t (1945) 495-

C. I:ROMA(;EOT .~XD P. [IHTZ, Mikrochem. Acta, 3 (r93 S) 52. [C P. HL;LLIN AND R. L. NOBLE, Biochem. J., 55 (t953) 289. E. I:. KOHLMILLER AND H. GEST, .[. [¢acteriol., 6~ (I05Ii 2(i(~. S. MORITA, J. 13iockem. Tokyo, 42 (r955) 533. K. A. DAWES, D. \V. RIB~¢ONS AND P. J. LARGE, I?iochcm../., 08 (I90O) 7%. I 4 T . [ 2. ['~ING ANI) r . H . (~HI~LDFLIN, J. l{lO[. Chem., 2o8 (I954) 82I. 15 J. I)ELI~v ,\ND J. S('m~LL, .l. Gen. 3licr(dfi~d., -'9 (r002) 589. o io Ir 12 13

Received June tzth, I96 7

t¢iockim. [4iopkyx. .4eta, t t8 (L967) 3o.t 3o(,