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Biosynthesis of vitamin K and ubiquinone relation to the shikimic acid pathway in Escherichia coli
204
P R E L I M I N A R Y NOTES
PN 2~ 042 Biosynthesis o f v i t a m i n
K and ubiquinone in Escherichia coii
Relation to the shikirnic acid pathway
The route of biosynthesis of the naphthoquinone "nucleus" of the vitamin K group of compounds is not known. Foliowing the observation z that acetate did net serve as a precursor for this "nucleus" in growing cells of Escherichi~ coil, tile main pathway for the synthesis of aromatic compounds in this organism, namely the shikimic acid pathway, was investigated. This has been done b y examining if [1~-C~shikimic acid was incorporated into vitamin K~. A method was deveioped for the extraction of both vitamin K and ubiquinore which avoided saponification. Subsequent thin-layer chromatography on silica gel gave discrete visible bands of each of these compounds. Vitamin K S was identified by its typical infrared absorption spectrum and ubiquinone b y its ultraviolet and infrared spectra. It has recently been shown that 4-hydroxybenzoic acid is incorporated into ubiquinone by rat kidney ~, Azotobacter ~, yeas~ 2, rat liver a and E. col#o During the present experiments shikimic acid was incorporated into ubiquinone and the addition of an excess of unlabelled 4-hydroxybenzoic acid eliminated this incorporation. [14C]Shikimic acid was also incorporated into vitamin K~ bat the addition of 5
Vitamin K
Ubiquinone
4
[~
:
Catechol
/T\
0/4
~
3[
/~
¢
(b) 4-hydroxyb . . . . ic
4,
4-
.:\ 3 i
2
3,4 -dlhydPoxybenza[dehyae
/
,.j' 0
(c) i :
0
2
_
~
,t
Distance
-~ i
6 from
8
10
solvent
12
=
fFont ,cm
F i g . i . T h e i n c o r p o r a t i o n of Z14C]shikimic a c i d i n t o v i t a m i n K 2 a n d n b i q u i n o n e . E f f e c t of c a t e c h o l , 4 - h y d r o x y b e n z o i c a c i d or 3 , 4 - d i h y d r o x y b e n z a l d e h y d e . C u i t u r e s of E . coli w e r e g r o w n for abon~c 6 h i n t h e f o l l o w i n g m e d i a : g l u c o s e c i t r a t e - m i n e r a l s a l t s m e d i u m p l u s Io -4 M L - p h e n y t a l a n i n e , x - t y r o s i n e a n d 5 - t r y p t o p h a n a n d 8.4 # g [ l ~ C ] s h i k i m i c a c i d (specific a c t i v i t y 8.4 m C m m o l e ] i n a t o t a l v o l u m e of 2 I. (a) p l u s zo -4 M c a t e c h o l ~ : (b] p l u s zo -4 29f 4 - h y d r o x y b e n z o i c a c i d ; (c) p l u s IO -~ M 3 , 4 - d i h y d r o x y b e n z a l d e h y d e . Cells w e r e e x t r a c t e d w i t h a c e t o n e w h i c h w a s r e m o v e d a n d the residue extracted with light petroleum. A concentrate was then chromatographed on silicagel t h i n - l a y e r p l a t e s w i t h c h l o r o f o r m - l i g h t p e t r o l e u m (8o:2o] a s s o l v e n t . A c t i v i t y : full s c a b d e f l e c t i o n : . . . . . . . , 3000 c o u n t s r a i n ; , Iooo counts Imim * Similar radiochromatograms or w i t h n o a d d i t i o n .
w e r e g i v e n b y s u b s t i t u t i n g v a r i o u s o t h e r c o m p o u n d s (see t e x t l
Biochgm. Biophys. Acta, 93 (z964) 2 o 4 - 2 o 6
PRELIMINARY NOTES
205
unlabelled 4-hydroxybenzoic acid did not affect the incorporation, indicating that the pathway from shikimic acid to vitamin K s did not involve 4-hydroxybenzoic acid. DAVIS~,5 showed that certain auxotrophs of E. coli which required for growth phenylalanine, tyrosine, tryptophan, 4-hydroxybenzoic acid and 4-aminobenzoic acid required another growth factor in a medium at pH 7.5. This "sixth factor" requirement could be satisfied by the addition of a number of four substituted pyrocatechol derivatives including 3,4-dihydroxybenzaldehyde and adrenaline. In the present experiments the addition of 3,4-dihydroxybenzaldehyde or adrenaline suppressed the incorporation of [~4C]shikimic acid into vitamin K but did not affect the incorporation into ubiquinone (Fig. I). This would suggest that sixth factor is related to vitamin K biosynthesis in E. coli. It has been suggested that 4-hydroxybenzoic acid for ubiquinone biosynthesis arises from 3-enolpyruvylshikimic acid 5-phosphate ~ but recent work would indicate that the most likely immediate precursor of 4-hydroxybenzoic acid is chorismic acids. Fig. 2 shows the most likely routes of biosynthesis of various aromatic compounds in E. coli and the evidence for sixth factor obtained with multiple aromatic auxotrophs would suggest that the branchpoint for vitamin K is at chorismic acid. However, more direct evidence is being sought using cell-free preparations, since chorismic acid and prephenic acid do not act as growth factors and presumably cannot enter bacterial cells. A number of other compounds were tested in similar experiments to those described in Fig. I. These included catechol, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, 2,3-dihydroxybenzoic acid and menadione. None of these compounds suppressed the incorporation of [14C]shikimic acid but it is possible that some of these cannot enter the bacterial cell.
s h i k i m i c acid
shikilnie acid 5 - p h o s p h a t e
[ 3 - e n o l p y r u v y l s h i k i m i c acid 5 - p h o s p h a t e
v i t a m i n t~Z2 <----- [ 3 , 4 - d i h y d r o x y - ] [benzaldehyde ]
<-----
c h o r i s m i c acid - - - + 4 - h y d r o x y b e n z o i c acid -----> u b i q u i n o n e
p r e p h e n i c acid
a n t h r a n i l i c acid
to p h e n y l a l a n i n e and tyrosine
to t r y p t o p h a n
4 - a m i n o b e n z o i c acid
to folic acid
Fig. 2. O u t l i n e of r o u t e s of b i o s y n t h e s i s of a r o m a t i c c o m p o u n d s in E. coll. B r o k e n arrows i n d i c a t e possible p a t h w a y s w h i c h h a v e n o t been e x a m i n e d a t t h e e n z y m i c level. Biochim. Biophys. Actc~, 93 (1964) 2 o 4 - 2 o 6
206
PRELIMINARY NOTES
We wish to thank Dr. M. G~ssox for 4-hydroxy [l~C]benzoic acid. This work was supported by grants from the Australian NationaI Health and )¢Iedical Research Council and the National Institute of Arthritis and Metabolic Diseases of the United States Public Hea!th Service (Grant AM-o4632 ).
Bacteriology School, U#iversity of Mdbour%e, Parkville, Victoria (Australia)
CTRAEME B. C o x ]?RANK G~BSO~-
1 G. ]3. Cox, u n p u b l i s h e d observations. z W. ~vV. PARSON AND H. RUDNEY, Proc. Nc~ll. Acad. Sci. U.S., 51 (I964) 444. a A. S. AIYAR AND R. 17;. OLSON, Federation P~'oc., 23 (~964) 425 . ]3. D. DAVIS, J. Bacteriol., 64 (I952) 729. B. D. DAvis, Cong~'. I~tern. Biockim., 2e, Paris, ~95& Syrup. 34etabo~sme ~/icrobie~< Masson, Paris, I952, p. 3 ~. M. I. G~BSON AND ~'. GIBSON, Biochem. J., 9o (~964) 248.
Received July 23rd, 1964 Bioehim. Biophys. Actc~, 93 (I964) 204-206
Px 2~. 049
Presence de I'acide I}-guanidoisobutyrique libre e~ combin6 chez des vers matins Au cours de l'isolement de l'hypotaurocyamine (acide 2-guanido{thane sutfinique), guanidine monosubstitu& biologique nouvelle de vers marins 1, nous avons constat6 la pr6sence, chez divers Sipunculiens, de guanidines monosubstitu6es inconnues dont nous avons entrepris l'6tude. L'un de ces corps, de caract~re tr~s alcalin, se zrouve en forte concentration dans le tractus de Phascolosoma vulgate Blainville, d'oh nous l'avons extrait, purifid par fractionnement sur rdsines &hangeuses d'ions et isold & !'6tat de picrate cristallis& Ce nouveau d~rivd, pour Requet nous proposons Ie nom de phascolosine, donne ]a r6action de Sakaguchi, reals ne rdagit avec aucun des autres rdactifs essav6s (ninhydrine, p-dim6thylaminobenzald6hyde, acide molybdique-SH2); par hvdrolvse acide, il lib~re une fraction amin& (r&ction de la ninhvdrine positive, r&ction de Sakaguchi n6gative), dont la structure est ~ l'6tude, et un constituant guanidique monosubstitu6 (r&ction de Sakaguchi positive, r6action de ia ninhvdrine ndgative]. qui a 6t6 isol6 & l'6tat de base libre cristallis6e. L'analyse 616mentaire du dernier produit correspond &la formule brute CsHl~O2Na qui est celle d'un acide guanidobutyrique (trouv6: C 40.9; H 7.7; O. 22.6; N. 29~o. Th6orie pour CaHI,02N8: C, 41.34; H. 7.58; O. 22.05; N. 28.94 ). I1 a 6t6 compar6 aux divers isom~res de l'acide guanidobutyrique, pr6par& par amidination des d6riv& amin6s correspondants selon SCld2TTE~: la chromatographie e, t'61ectrophor~se sur papier dans plusieurs m61anges de solvants qui donnent une bonne r6solutioll des isom~res ~-, f3-et 7-, l'identifiaient 5 un acide /~-guanidobutyrique, reals les Isom~res et iso ne se sdparaienl darts aucun des solvants essav6s. La d6gradation bary~ique de ce composd a conduit ~ !a formation de l'acide ~-aminobutynque correspondant. Biochim. Biophys. AcZa, 93 (1964) 206-208
P R E L I M I N A R Y NOTES
PN 2~ 042 Biosynthesis o f v i t a m i n
K and ubiquinone in Escherichia coii
Relation to the shikirnic acid pathway
The route of biosynthesis of the naphthoquinone "nucleus" of the vitamin K group of compounds is not known. Foliowing the observation z that acetate did net serve as a precursor for this "nucleus" in growing cells of Escherichi~ coil, tile main pathway for the synthesis of aromatic compounds in this organism, namely the shikimic acid pathway, was investigated. This has been done b y examining if [1~-C~shikimic acid was incorporated into vitamin K~. A method was deveioped for the extraction of both vitamin K and ubiquinore which avoided saponification. Subsequent thin-layer chromatography on silica gel gave discrete visible bands of each of these compounds. Vitamin K S was identified by its typical infrared absorption spectrum and ubiquinone b y its ultraviolet and infrared spectra. It has recently been shown that 4-hydroxybenzoic acid is incorporated into ubiquinone by rat kidney ~, Azotobacter ~, yeas~ 2, rat liver a and E. col#o During the present experiments shikimic acid was incorporated into ubiquinone and the addition of an excess of unlabelled 4-hydroxybenzoic acid eliminated this incorporation. [14C]Shikimic acid was also incorporated into vitamin K~ bat the addition of 5
Vitamin K
Ubiquinone
4
[~
:
Catechol
/T\
0/4
~
3[
/~
¢
(b) 4-hydroxyb . . . . ic
4,
4-
.:\ 3 i
2
3,4 -dlhydPoxybenza[dehyae
/
,.j' 0
(c) i :
0
2
_
~
,t
Distance
-~ i
6 from
8
10
solvent
12
=
fFont ,cm
F i g . i . T h e i n c o r p o r a t i o n of Z14C]shikimic a c i d i n t o v i t a m i n K 2 a n d n b i q u i n o n e . E f f e c t of c a t e c h o l , 4 - h y d r o x y b e n z o i c a c i d or 3 , 4 - d i h y d r o x y b e n z a l d e h y d e . C u i t u r e s of E . coli w e r e g r o w n for abon~c 6 h i n t h e f o l l o w i n g m e d i a : g l u c o s e c i t r a t e - m i n e r a l s a l t s m e d i u m p l u s Io -4 M L - p h e n y t a l a n i n e , x - t y r o s i n e a n d 5 - t r y p t o p h a n a n d 8.4 # g [ l ~ C ] s h i k i m i c a c i d (specific a c t i v i t y 8.4 m C m m o l e ] i n a t o t a l v o l u m e of 2 I. (a) p l u s zo -4 M c a t e c h o l ~ : (b] p l u s zo -4 29f 4 - h y d r o x y b e n z o i c a c i d ; (c) p l u s IO -~ M 3 , 4 - d i h y d r o x y b e n z a l d e h y d e . Cells w e r e e x t r a c t e d w i t h a c e t o n e w h i c h w a s r e m o v e d a n d the residue extracted with light petroleum. A concentrate was then chromatographed on silicagel t h i n - l a y e r p l a t e s w i t h c h l o r o f o r m - l i g h t p e t r o l e u m (8o:2o] a s s o l v e n t . A c t i v i t y : full s c a b d e f l e c t i o n : . . . . . . . , 3000 c o u n t s r a i n ; , Iooo counts Imim * Similar radiochromatograms or w i t h n o a d d i t i o n .
w e r e g i v e n b y s u b s t i t u t i n g v a r i o u s o t h e r c o m p o u n d s (see t e x t l
Biochgm. Biophys. Acta, 93 (z964) 2 o 4 - 2 o 6
PRELIMINARY NOTES
205
unlabelled 4-hydroxybenzoic acid did not affect the incorporation, indicating that the pathway from shikimic acid to vitamin K s did not involve 4-hydroxybenzoic acid. DAVIS~,5 showed that certain auxotrophs of E. coli which required for growth phenylalanine, tyrosine, tryptophan, 4-hydroxybenzoic acid and 4-aminobenzoic acid required another growth factor in a medium at pH 7.5. This "sixth factor" requirement could be satisfied by the addition of a number of four substituted pyrocatechol derivatives including 3,4-dihydroxybenzaldehyde and adrenaline. In the present experiments the addition of 3,4-dihydroxybenzaldehyde or adrenaline suppressed the incorporation of [~4C]shikimic acid into vitamin K but did not affect the incorporation into ubiquinone (Fig. I). This would suggest that sixth factor is related to vitamin K biosynthesis in E. coli. It has been suggested that 4-hydroxybenzoic acid for ubiquinone biosynthesis arises from 3-enolpyruvylshikimic acid 5-phosphate ~ but recent work would indicate that the most likely immediate precursor of 4-hydroxybenzoic acid is chorismic acids. Fig. 2 shows the most likely routes of biosynthesis of various aromatic compounds in E. coli and the evidence for sixth factor obtained with multiple aromatic auxotrophs would suggest that the branchpoint for vitamin K is at chorismic acid. However, more direct evidence is being sought using cell-free preparations, since chorismic acid and prephenic acid do not act as growth factors and presumably cannot enter bacterial cells. A number of other compounds were tested in similar experiments to those described in Fig. I. These included catechol, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, 2,3-dihydroxybenzoic acid and menadione. None of these compounds suppressed the incorporation of [14C]shikimic acid but it is possible that some of these cannot enter the bacterial cell.
s h i k i m i c acid
shikilnie acid 5 - p h o s p h a t e
[ 3 - e n o l p y r u v y l s h i k i m i c acid 5 - p h o s p h a t e
v i t a m i n t~Z2 <----- [ 3 , 4 - d i h y d r o x y - ] [benzaldehyde ]
<-----
c h o r i s m i c acid - - - + 4 - h y d r o x y b e n z o i c acid -----> u b i q u i n o n e
p r e p h e n i c acid
a n t h r a n i l i c acid
to p h e n y l a l a n i n e and tyrosine
to t r y p t o p h a n
4 - a m i n o b e n z o i c acid
to folic acid
Fig. 2. O u t l i n e of r o u t e s of b i o s y n t h e s i s of a r o m a t i c c o m p o u n d s in E. coll. B r o k e n arrows i n d i c a t e possible p a t h w a y s w h i c h h a v e n o t been e x a m i n e d a t t h e e n z y m i c level. Biochim. Biophys. Actc~, 93 (1964) 2 o 4 - 2 o 6
206
PRELIMINARY NOTES
We wish to thank Dr. M. G~ssox for 4-hydroxy [l~C]benzoic acid. This work was supported by grants from the Australian NationaI Health and )¢Iedical Research Council and the National Institute of Arthritis and Metabolic Diseases of the United States Public Hea!th Service (Grant AM-o4632 ).
Bacteriology School, U#iversity of Mdbour%e, Parkville, Victoria (Australia)
CTRAEME B. C o x ]?RANK G~BSO~-
1 G. ]3. Cox, u n p u b l i s h e d observations. z W. ~vV. PARSON AND H. RUDNEY, Proc. Nc~ll. Acad. Sci. U.S., 51 (I964) 444. a A. S. AIYAR AND R. 17;. OLSON, Federation P~'oc., 23 (~964) 425 . ]3. D. DAVIS, J. Bacteriol., 64 (I952) 729. B. D. DAvis, Cong~'. I~tern. Biockim., 2e, Paris, ~95& Syrup. 34etabo~sme ~/icrobie~< Masson, Paris, I952, p. 3 ~. M. I. G~BSON AND ~'. GIBSON, Biochem. J., 9o (~964) 248.
Received July 23rd, 1964 Bioehim. Biophys. Actc~, 93 (I964) 204-206
Px 2~. 049
Presence de I'acide I}-guanidoisobutyrique libre e~ combin6 chez des vers matins Au cours de l'isolement de l'hypotaurocyamine (acide 2-guanido{thane sutfinique), guanidine monosubstitu& biologique nouvelle de vers marins 1, nous avons constat6 la pr6sence, chez divers Sipunculiens, de guanidines monosubstitu6es inconnues dont nous avons entrepris l'6tude. L'un de ces corps, de caract~re tr~s alcalin, se zrouve en forte concentration dans le tractus de Phascolosoma vulgate Blainville, d'oh nous l'avons extrait, purifid par fractionnement sur rdsines &hangeuses d'ions et isold & !'6tat de picrate cristallis& Ce nouveau d~rivd, pour Requet nous proposons Ie nom de phascolosine, donne ]a r6action de Sakaguchi, reals ne rdagit avec aucun des autres rdactifs essav6s (ninhydrine, p-dim6thylaminobenzald6hyde, acide molybdique-SH2); par hvdrolvse acide, il lib~re une fraction amin& (r&ction de la ninhvdrine positive, r&ction de Sakaguchi n6gative), dont la structure est ~ l'6tude, et un constituant guanidique monosubstitu6 (r&ction de Sakaguchi positive, r6action de ia ninhvdrine ndgative]. qui a 6t6 isol6 & l'6tat de base libre cristallis6e. L'analyse 616mentaire du dernier produit correspond &la formule brute CsHl~O2Na qui est celle d'un acide guanidobutyrique (trouv6: C 40.9; H 7.7; O. 22.6; N. 29~o. Th6orie pour CaHI,02N8: C, 41.34; H. 7.58; O. 22.05; N. 28.94 ). I1 a 6t6 compar6 aux divers isom~res de l'acide guanidobutyrique, pr6par& par amidination des d6riv& amin6s correspondants selon SCld2TTE~: la chromatographie e, t'61ectrophor~se sur papier dans plusieurs m61anges de solvants qui donnent une bonne r6solutioll des isom~res ~-, f3-et 7-, l'identifiaient 5 un acide /~-guanidobutyrique, reals les Isom~res et iso ne se sdparaienl darts aucun des solvants essav6s. La d6gradation bary~ique de ce composd a conduit ~ !a formation de l'acide ~-aminobutynque correspondant. Biochim. Biophys. AcZa, 93 (1964) 206-208