Studies on the microbial formation of sugar derivatives of D -pantothenic acid

91

B I O C H I M I C A ET B I O P H Y S I C A ACTA

BBA

26975

S T U D I E S ON T H E MICROBIAL FORMATION OF SUGAR D E R I V A T I V E S OF D - P A N T O T H E N I C ACID I. I S O L A T I O N AND I D E N T I F I C A T I O N OF A N E W D E R I V A T I V E OF PANTOT H E N I C ACID: 4'(2')-O-(~-D-GLUCOPYRANOSYL)-D-PANTOTHENIC ACID

FUSAKO

K A W A I ~, K E N S E I

M A E Z A T O n, H I D E A K I

Y A M A I ) A b AND K O I C H I

()GATA a

a Department of Agricult~ral Chemistry, tqyoto University, tfyoto 606 (Japan) a~d t, l?cs~arch Institute for Food Science, Kyoto University, U/i, Kyoto 6zz (Japan) ( R e c e i v e d J u l y 3rd, i 9 7 e)

SUMMARY

A new derivative of pantothenic acid was found in a reaction mixture of

Sporobolo~©,ces coralliformis IFO lO32 incubated in a medium containing maltose and D-pantothenic acid. This new compound was isolated from an incubation mixture by active charcoal, Amberlite CG-5o, Sephadex G-Io and DEAE-cellulose column chromatography, and by paper chromatography. Then, the compound was characterized as 4'(2')-0-(e-D-glucopyranosyl)-D-pantothenic acid by various analytical methods, including bioautography, paper chromatography and infrared and NMR spectroscopy.

INTRODUCTION

Pantothenic acid is widely distributed in animals, plants and microorganisms as a component of CoA and plays an important role in m a n y metabolic pathways. Many workers have investigated the biosynthetic and biodegradative metabolism of pantothenic acid. A large number of derivatives of pantothenic acid have been found in nature and have also been chemically sythesized. Recently, a fl-glucoside of Dpantothenic acid was isolated from tomato juice as a growth factor for malo-lactic fermentation bacteria~, 2. The compound showed a microbial activity IOO times as great as free D-pantothenic acid for a certain lactic acid bacterium. However, an ~-glucoside of pantothenic acid has been neither chemically nor biologically synthesized. The present communication describes the microbial formation of tile y.-glucoside of D-pantothenic acid, its isolation, and its characterization. EXPERIMENTAL

Chemicals DEAE-cellulose was kindly given by the Green Cross Co., Osaka. Biochirn. Biophys. Acta, 286 (1972) 9 1 - 9 7

92

F. KAWAI 63t a[.

Other chemicals used in this study were obtained from commercial sources. Cellulase (Type II, from Aspergillus niger, Sigma Chem. Co.), fl-glucosidase (fronl almond, Sigma Chem. Co.), maltase (Grade II, from A. niger, Sigma Chem. Co.) and u.-glucosidase (from yeast, Boehringer Mannheim Japan K.K.) were also purchased through commercial routes.

Microorganism and cultivation SporobolonLvces coralhformis IVO lO32 was grown on a medium containing 5 .0 o/ o glucose, 0.5% peptone, o -o =o/ 'o .yeast extract, o.5% dibasic potassium phosphate, o.2% monobasic potassium phosphate, and o.o2°.o MgSO4, adjusted to pH 6.o. The cultivation was carried out for 48 h at 28 °C with 5o ml of the medium in a 3oo ml shaking flask on a reciprocal shaker. After cultivation, the cells were collected by continuous centrifugation at 5ooo rev./min and washed once with o.oI M potassium phosphate buffer (pH 7.o) containing 5 .to a M 2-mercaptoethanol and suspended in 5 ml of the same buffer. This suspension was used as the intact cell preparation, following the reaction.

Reaction with intact cells of S. coralliformis The standard reaction mixture contained 5° hmoles of D-pantothenic acid, IOO }elnoles of maltose, IOO/ramies of potassium phosphate buffer (pH 7.o), 3//moles of 2-mercaptoethanol, a final concentration of o . I % of cetyltrimethylanm~onium bromide, and o.5 ml of intact cell suspension in a total volume of 1. 5 ml. Glucose was used in place of maltose as the blank. The incubation was made for 24 h at 28 °C with shaking. The reaction was stopped by immersing the tube in a boiling water bath for 5 min. The supernatant fluid obtained by eentrifugation was assayed for a derivative of D-pantothenic acid.

A Hal),s£s A derivative of pantothenic acid was identified by a bioautographic technique as follows. A suitable amount of the supernatant fluid was spotted on Toyo tilter paper No. 53 (2 cm in width) and developed with a solvent system of J>butanolacetic acid-water (4: I : i , by vol.). The paper strip was cut off in o.5 cm widths from the e cm width followed by bioautography, using Saccharoncvces carlsbergcl~sis ATCC 9o8o (ref. 3), which has a growth requirement for pantothenic acid in the presence of asparagine. Pantothenic acid was quantitatively bioassayed by the paper dis(: method, using S. carlsbcrgensis ATCC 9080 (ref. 3). Paper chromatography of sugars was carried out with the solvent systems described in Table II and Fig. I, and the AgNOa reagent ~ was sprayed for sugar detection. Glucose was determined by the glucostat method ~ and reducing sugar was estimated by the Somogyi-Nelson method%

Determination of infrared and A:MR spectra Infrared spectra were measured in a micro KBr tablet with a Hitachi infrared spectrometer type EPI-G3. NMR spectra were determined on a \:arian HA-IooD spectrometer at xoo MHz in deuterium oxide with [~Hs]DSS (sodium 2,2-dimethyl-2silapentane-5-sulfonate) as an internal standard.

Biochim. Biophys. Acta, 286 (1972) 9I 97

93

A NEVV D E R I V A T I V E OF P A N T O T H E N I C ACID

RESULTS

The formation of a glucoside of D-pantothenic acid from maltose and D-pantothenic acid was investigated with various microorganisms. Screening showed that a remarkable amount of the derivative was formed in a reaction mixture of S. coralliformis I1;O lO32 containing maltose and D-pantothenic acid. The derivative was not formed in a glucose medium. Tile isolation and identification of the compound were attempted as follows. Isolation of a new derivative of pantothenic acid S. coralhformis IFO lO32 was grown on a medium containing maltose o/ ,,o peptone o.5°,o, yeast extract o.5%, dibasic potassium ptaosphate o . 5 ° monobasic potassium phosphate o.2°/o, and 3'lgSO~ o.o2°/o, adjusted to pH 6.0. Cultivation was made for 48 h at 28 °C with 500 ml of the medium in a 2-I shaking flask on a reciprocal shaker. The cells were collected by continuous centrifugation at 5ooo rev./min, washed once with 0.9% saline solution, and suspended in the same solution. 21 g (dry wt) of intact cells of tim organism were incubated aerobically for 4 ° h at 28 °C with 4 ° g of maltose, 4 g of D-pantothenic acid, 8 mmoles of potassium phosphate buffer (pH 7.o), and o. 4 g of cetyl trimethyl ammonium bromide in a final volume of 4oo ml. The reaction was stopped by immersing the incubation mixture (adjusted to pH 7.o) in boiling water for 5 min, then the cells were removed by centrifugation at IOOOO rev./min for 2o rain. The supernatant solution was adjusted to pH 4.o and was left at 4 °C overnight. Any material precipitating was removed by filtration. Active charcoal (6o g) was added to the filtrate. After stirring the filtrate for I h, the charcoal was separated, washed with o.oI M HC1 and water, and eluted with five volumes of acetone. The eluate was evaporated in vacuo to a small volume. The concentrated solution was subjected to paper chromatography with Toyo filter paper No. 526 in a solvent system of n-butanol acetic acid-water (4:1:1, by vol.) and bioautography was carried out with S. carlsbergensis ATCC 9o8o (ref. 3)- The active zone, which differed from that of D-pantothenic acid, was extracted with j~o°/,,o ethanol. The extract was concentrated in vacuo and desalted with Amberlite CG-5o (H ~ type). The concentrated effluent was applied to a Sephadex G-io column (I cm x lO5 cm) and eluted with water. Active fractions were adsorbed on DEAE-cellulose (OH type), then were eluted with o.o2 M LiC1. The eluted fraction was desalted with Amberlite CG-5o ( H type), TABLE

I

/ ~ F VALUES FROM B I O A U T O G R A P H Y

l , n - b u t a n o l a c e t i c a c i d - w a t e r (4: i : i , b y vol.) ; 1I, ~z-butanol p y r i d i n c w a t e r (6: 4: 3, b y vol.) ; I I I , n - p r o p a n o l - 2 8 ° o N H a O H w a t e r (6 : 3 : i, b y vol.) ; I V, n - b u t a n o l e t h a n o l - w a t e r (5 : i : 4, b v VOI., u p p e r l a y e r ) ; V, i s o b u t y r i c a c i d o.5 M N H 4 O H o. I }] E D T A (~ o o : S o : 1.6, b y vol).

Compom~d ~-Glucosyl-D-pantothenic acid D-pantothenic acid /3-Alanine 4 -Phosphopantothenic acid 7 Panthenol Pantethine Pantoyl lactone

(IgF value in Solvent) I II Ill o.49 0.36 o.76 o.5o o.29 o.13 o.45 o. 19 No response No response No response

0.63 o.76 o.54 o.28

11....

1"

0.52 o.85 o.39 o.4 °

0.48 o.69 o.62 o.38

Biochim. Biophys. Acla, 286 ( i 9 7 2 ) 9 1 - 9 7

94

F. KAWAI 6t a].

TAB1A'; I[ IDENTIFICATION

OF

PANTOTHENIC

ACID

ANI)

GLI:TCOSE

MOIETIES

OF

THF

ISOLATED

COMPOUND

The p a n t o t h e n i c acid moiety was con lirmed b y b i o a u t o g r a p h y using Sacckaromyces carlsbergensis ATCC 9o8o and the glucose moiety was identified by the color
RF vahws i~ Solwwt I ll

Isolated conlpound Acid h y d r o l y z a t c * Enzvmatic hydrolyzate* * 1~an{othenic acid /3-Alanine Glucose Maltose

o.40 o. So o.So o. 79 o.3-'

o.58 o. 76 o.73 o. 75 o.55

--

Ill

II"

0.34 0.33 o.3~

0.7.3 0.57 o.55

0.33 o. _,2

0.57 o. 49

* l l y d r o l y z e d in 0.0275 M H2SO ~ at l 2o 'C and i 5 lb for 3h. ** H y d r o l y z e d b y yeast c~-glucosidase.

a d j u s t e d t o p H 5.0 w i t h i 5I H ( ' I . S a t u r a t e d

Ba(OH)2 solution was added to the frac-

t i o n t o adjust t h e p H t o 7.o 7.2. E x c e s s Ba(OH),., w a s r e m o v e d r e p e a t e d l y b y c o n c e n t r a t i o n a n d f i l t r a t i o n . T h e c o m p l e t e l y d r i e d b a r i u m s a l t of t h e c o m p o u n d w a s d i s s o l v e d in m e t h a n o l . A f l u f f y p r e c i p i t a t e w a s o b t a i n e d b y a d d i n g e t h e r . T h e p r e c i p i t a t e w a s d i s s o l v e d in w a t e r a n d s u b j e c t e d t o p a p e r c h r o m a t o g r a p h y using Toyo filter paper N o . 53 in t h e s o l v e n t s y s t e m d e s c r i b e d a b o v e . T h e a c t i v e z o n e w a s e x t r a c t e d w i t h 5o°{) e t h a n o l , e v a p o r a t e d , l y o p h i l i z e d , a n d d r i e d i n vacuo o n P o e ; a n d K O H 21. 9 nag a s t h e b a r i u m s a l t ) .

(yield

S
~0.5

a

b

c

d

e

Authenti(

20.5 g]ucose

CD Maltose a

b

c

d

e

A u ~ h e n t ic

i:ig. I. Enzymatic hydrolysis of the isolated c o m p o u n d

by glucosidases. The pantothenie acid

moiety was contirmed b y b i o a u t o g r a p h y and the glucose moiety was d e t e r m i n e d 1)y the color d e v e l o p n l c n t with the AgNO a reagent. PaA, p a n t o t h e n i c acid; []-Ala, [~-alanine; tr., trace, a, mold maltase; b, yeast ~-glucosidase; c, /J glucosidase (emulsin); d, cellulase; e, isolated compound. Solvents: l, n-lmtauol-acetic acid water (4: ~ : i, b y vol.) I[, n-butanol -benzene pyridine water (5:1:3:3, byvol.). Biochim. Biopkys. Acta, 286 (tO72) 91-97

95

A NEW DERIVATIVE OF PANTOTHENIC ACID 2.5am

3

4

5

6

7

8

9

10

15

20

25

I00

E

4ooo Woo 3o?o 2+;o 2o~o '18;o 'l+'oo 'lloo '12;o 'le'oo

'

8;o

'

+;o

'

400

Wave number(cm -1 )

Fig. 2. The infrared spectrum of the isolated compound. ]'he spectrum was recorded on a Hitachi infrared spectrometer type EPI-G3 in a micro KBr tablet. The lyophilized p o w d e r w a s u s e d pound.

for the f u r t h e r c h a r a c t e r i z a t i o n of the con>

Identification of a derivative of pantothenic acid W i t h the isolated c o m p o u n d , t h e identification of the moieties of p a n t o t h e n i c acid a n d glucose was p e r f o r m e d a n d the s t r u c t u r e of the c o m p o u n d was suggested. RF values on b i o a u t o g r a p h y in various solvent s y s t e m s were different from the k n o w n m e t a b o l i t e s of p a n t o t h e n i c acid, as shown in Table I. The acid a n d e n z y m a t i c h y d r o l y s i s released p a n t o t h e n i c acid a n d glucose on the b i o a u t o g r a m a n d p a p e r c h r o m a t o g r a m , as shown in Table If. The m o l a r ratio of the two c o m p o n e n t s was I : I as d e t e r m i n e d b v the g l u c o s t a t m e t h o d s a n d b y bioassay. No reducing a c t i v i t y of the isolated c o m p o u n d was found using t h e S o m o g y i - N e l s o n m e t h o d < The e n z y m a t i c h y d r o l y s i s of the c o m p o u n d was i n v e s t i g a t e d to d e t e r m i n e t h e t y p e of glucosidic linkage. The result is shown in Fig. I. The c o m p o u n d was h y d r o l y z e d b y y e a s t a n d m o l d maltases, b u t not b y cellulase a n d fl-glucosidase. These d a t a suggested t h a t the c o m p o u n d was a - g l u c o p y r a n o s y l - o - p a n t o t h e n i c acid. The infrared s p e c t r u m s u p p o r t e d CILOH

-CH,ctcH,,],CR(0H)COXHCH.',CH,C00H

1t

0H CH,OH o }l 1 C(CHI)~CH,OH

~6) ~ L - - - - - / 1 - O -c. [ OH

** * CONHCH:CHaC00H

- C (CII v

,-

2LHiO

- C H 2- a n d r i n g p r o t o n s of g l u c o s e

-CIt2 -

_CH 2-

I

5.0

L

a.0

i

3.0

2.0

i

1.0

i

i

0

6 ppm

Fig. 3. T h e N M R s p e c t r u m of t h e isolated c o m p o u n d . T h e spectruna was recorded on a V a r i a n H : \ - l o o D s p e c t r o m e t e r a t i o o M H z in d e u t e r i u m o x i d e w i t h 12H~]DSS as an i n t e r n a l s t a n d a r d .

Biochim. Biophys. Acta, 286 (197 z) 91-97

96

1:. KAWAI ct a[.

this (Fig. 2). The fine structure was confirmed using the NMR spectrum, as shown in Fig. 3. Examination of the NMR spectrum (in ~'H,O, d ppm) showed two methyl signals (C-3', o.98 and o.99, 6H, s), a methylene signal of ('-2 (2.42, 2H, t, j 13 Hz), a methylene signal of C-3 (3-45, 2H, t, j 11.5 Hz), and a methylene signal of C- 4' and ring protons of the sugar moiety (3.55 3.9 o, m ) , a methvne signal ((;-2', 4.12, zH, s), and one anomeric proton signal of the ~-glucosidic linkage (4.89, i It, d, j - 3-5 ttz). Comparison of this spectrum with that of pantothenic acid suggests that the pantothenic acid moiety is contained in this compound. Comparison of the integral wtlues of the two methyl groups of the t)antothenic acid residue with that of ¢) 3-55 3.9 o ppm showed that the ratio of the pantothenic acid and glucose moieties was approximately one to one. The proposed structures for the compound are shown in Fig. 4. Comtmring

CH2OH

o#O OH

H

I

0 -- CH2C(CH3)2CFt(OH)CONHCH2CH2COOH

OH 4'-- 0 I(~--o--GlucopyranosyDID--pantothenic acid (I) CH2OH O H ~ O'x

HO[~

C(CHa)2CH2OH

' ~ O--~H OH CONHCH2CH2COOH

2'--O--(a--D--Glucopycanosyl)--D--pantothenic acid (1Z) Fig. 4. P r o p o s e d s t r u c t u r e s for c~-glucosyl-D t)antothenic acid.

the methyne signal (d 4.12 ppm) of ~-D-glucopyranosyl-D-pantothenic acid with the signals of the 2 ' - O - ( f l - D - g l u c o p y r a n o s y l ) - D - p a n t o t h e n i c acid and 4'-0-(fl-D-glucopyranosyl)-D-pantothenic acid reported by Amaehi el al.1, "- suggested that the glucosidic linkage might be present at C-4'. Thus, this compound contains one mole of glucose and one mole of pantothenic acid, probably conjugated as 4'-O-(a-D-glucopyranosyl)-bpantothenic acid. DISCUSSION

The formation of riboflavinyl-a-glucoside bv rat liver was first flmnd by \'Vhitby in I95OL Katagiri et al. 9- 11 investigated the microbial formation of various riboflavinvlglycosides, including riboflavinyl-glucoside, -galaetoside, -fructoside, and various oligosaccharides. Suzuki and Uchida also reported the formation of riboflavinylglycosides bv microorganisms and enzyme preparations prepared from microorganisms and plants1~, ~a. We found the formation of pyridoxine glucoside in microorganisms14, is. Miyake et a l ? ~ reported the enzymatic formation of the sugar derivative of ascorbic acid. Although the physiological role of the sugar derivatives of various vitamins is still not clear, it is significant that various sugar derivatives naturally occur. It was suggested that the activity of the fi-glucoside of D-pantothenic acid was due to the specific membrane transport of malo-lactic fermentation bacteria '2. Recently, ribofla]~iochim. l~h)phys. Acfa, 286 (i97 z) 91 97

A NEW DERIVATIVE OF PANTOTHENIC ACID

97

v i n y l - ~ - g l u c o s i d e was e x t r a c t e d f r o m cat liver~L T h e s e f a c t s m a y h a v e a b e a r i n g on t h e p h y s i o l o g i c a l significance of t h e s e c o m p o u n d s . T h e a u t h o r i s o l a t e d a n e w d e r i v a t i v e of n - p a n t o t h e n i c a c i d f r o m a r e a c t i o n m i x t u r e of S. coralliformis. I t was s u g g e s t e d t h a t t h e g l u c o s i d i c l i n k a g e of t h e c o m p o u n d m i g h t be p r e s e n t at C- 4' f r o m t h e c o m p a r i s o n w i t h t h e 4'- or 2'-fi-glucoside of D - p a n t o t h e n i c a c i d a n d t h e f a c i l i t y of r e a c t i v i t y of t h e C-4' or C-2' alcoholic groups. I t r e m a i n s u n c l a r i f i e d w h e t h e r t h e c o m p o u n d p l a y s t h e s a m e p h y i s o l o g i c a l role as t h e fi-glucoside of D - p a n t o t h e n i c a c i d or not. ACKNOWI.EDGEMENTS W e wish to e x p r e s s o u r sincere t h a n k s to D r T e t s u r o S h i n g u , t h e F a c u l t y of P h a r m a c o l o g y , a n d to D r T a m i o U e n o , t h e P e s t i c i d e R e s e a r c h I n s t i t u t e , K y o t o U n i v e r s i t y , for t h e i r k i n d a s s i s t a n c e in m e a s u r i n g a n d i n t e r p r e t i n g t h e N M R s p e c t r a . T h a n k s are also d u e to t h e L a b o r a t o r y of A n a l y s i s of A g r i c u l t u r a l P r o d u c t s , D e p a r t m e n t of F o o d Science a n d T e c h n o l o g y , K y o t o U n i v e r s i t y , for t h e a s s i s t a n c e in m e a s u r i n g t h e i n f r a r e d a n d N M R s p e c t r a . REFERENCES i Ainachi, T., lmamoto, S., Yoshizumi, H. anti Senoh, S. (I97 o) Tetrahedron Letters, 487 f 2 Amachi, T., hnamoto, S. and Yoshizumi, H. (i97 I) Agr. Biol. Chem. 35, i22z 3 Atkm, L., Williams, \V. L., Schultz, A. S. and Frey, C. N. (~944) Ind. Eng. Chem., Anal. ed. I6, 67 4 Trevelyan, \V. E., Procter, D. P. and Harrison, J. S. (I95 o) Nature, i66, 444 5 Sols, A. and de la Fuente, G. (I96I) 3Iethods in 3]edical Research, Vol. 9, Year book Publishers, Chicago 6 Somogyi, M. (~952) J. Biol. Chem. I95, ~9 7 Ogata, K., Shimizu, S. and Tani, Y. (I97 t) Agr. Biol. Chem. 36, 84 8 Whitby, L. G. (i95 o) Nature i66, 479 9 l(atagiri, H. and Tachibana, S. (I953) Vitamins, Kyoto 6, 674 io Katagari, H. and Tachibana, S. (~953) Vitamins, t(yoto 6, 842 i i Katagiri, H., Yamada, H. and hnai, K. (I957) J. Vitaminol. 3,264 i2 Suzuki, Y. and Uchida, K. (i97 o) Vitamins, Ifyoto 4 I, 338 13 Suzuki, Y. and Uchidag K. (i97 o) J. Agr. Chem. Soe..lap. 44, I57 14 Ogata, ix2., Tani, Y., Uchida, Y. and Tochikura, T. 0968) Biochim. Biophys. Acta I65, 578. 15 Tani, Y., Kawai, F., Uchida, Y., Tochikura, T. and Ogata, K. (I969) J. Vitaminol. I5, I67 I6 Miyake, T. and Suzuki, Y. (t97 I) Vitamins, Ifyoto 43, 205 17 Kasai, S., Isemura, S., Masuoka, M. and Matsui, K. (I972) J. Vitaminol. i8, 17 Biochim. Biophys. dcta, 286 (i972) 9I 97