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Biosynthesis of the glucuronic acid unit of hemicellulose B from UDP-glucuronic acid
572
SHORT
COMMUNICATIONS
BBA 2337 I
Biosynthesis of the glucuronic acid unit of hemicellulose B from UDP-glucuronic acid The term hemicellulose designates a chemically ill-defined group of plant cellwall heteropolysaccharides, which may be composed of different monosaccharides with various types of linkages. A feature in common seems to be the occurrence of single molecule side chains of glucuronic acid or 4-0-methylglucuronic acid, the proportion of the uronic acid being higher in the hemicellulose B fraction than in the hemicellulose A fraction I 4. It was recently shown for the 4-0-methyl-glucurono-xylan from corn cobs that the 4-0-methyl ether group is introduced into preformed polysaccharide molecules s. Experiments on the biosynthesis of the glucuronic acid unit of hemicellulose B are reported here. They indicate that the particulate enzyme preparation from immature corn cobs contains, in addition to the methyl transferase, an enzyme which introduces the glucuronic acid group from UDP-glucuronic acid into hemicellulose B. All materials, operations and methods for the analysis of the polysaccharides are essentially the same as in the study on the formation of the methyl ether s . Labelled UDP-glucuronic acid was prepared by enzymatic dehydrogenation of UDP-glucose a. 6-/3-Glucuronosyl-galactose was a gift from Dr. G. O. ASPINALL. TABLE I LABELLED PRODUCTS FORMED FROM
UDP-I14C]GLUCURONIC
RATIONS
ABSENCE
FROM
CORN
COBS IN
THE
AND
ACID BY PARTICULATE ENZYME PREPA-
PRESENCE
OF
UDp-F12C]XYLOSF.
The incubation m i x t u r e s contained 2oo/~1 enzyme preparation, 2o0 ill p h o s p h a t e buffer (o.i M, p H 6.3), i ~o albumin, o. 4 M sucrose, IOO m/,moles MgC12, 6.8 nil, moles (696ooo counts/mini UDP-I14C~]glucuronic acid and no, or 12o ml, moles U D P - I I ~ Q x y l o s e (final p H 6.5). The m i x t u r e was incubated at 3 °0 for 15 min and the reaction stopped by addition of 2 ml boiling 99 ~o ethanol. The material was washed twice with 2 ml 75 % ethanol, extracted twice with i ml of o. 5 % amm o n i u m oxalate a n d t h e n with 2 ml of 2 ~o N a O H , each step with heating to 80 ° for 5 min. The p o l y s a c c h a r i d e s were precipitated from the a m m o n i u m oxalate solution by addition of 5 vol. 99 ~o ethanol. The N a O H solution was b r o u g h t to p H 4.7 with acetic acid, the precipitate (hemicellulose A) collected and t h e n t h e h e m i c e l l u l o s e B fraction precipitated by addition of 5 vol. 99 °/o ethanol.
Incorporation inlo polysaccharides
. UDP-[aeCqXyl (counts/rain "z ±o a)
+ UDP-[Iz2QA'y! (counts/rain >4 ±o a)
Total Soluble in a m m o n i u m oxalate (total) Precipitated from a n l m o n i u m oxalate Acid hydrolysate of ppt. from a m m o n i u n l oxalate: neutral sugars uronic acids Soluble in N a O H (total) : helnicellulose A hemicellulose B Acid hydrolysis of h e m i e e l l u l o s e B: n e u t r a l sugars uronic acids
395 23o I76"
03 35 22
129. 4 2. 3 165 62 IOO
12.6 3.8 29 i2 15
76.8 4. i
7.1 4.8
* 9o °dialysable aud t h e r e f o r e m o s t likely represents short-chain p o l y s a c c h a r i d e s .
Biochim. Biophys. Acta, I,i8 (1967) 572-574
SHORT COMMUNICATIONS
573
On incubation of the particulate enzyme preparation with UDP-E14Ce]glucuronic acid, more than half of the radioactivity was incorporated into polysaccharides (Table I). Fractional elution and precipitation, together with acid hydrolysis and paper chromatography, were used to characterize these products. An appreciable part of the material was solubilized with a solution of ammonium oxalate. Although the proportion of uronic acids in this fraction was smaller than in the hemicellulose B fraction, essentially the same radioactive sugars and uronic acids as in hemicellulose B were present. The hemicellulose B fraction was characterized in greater detail by acid hydrolysis and separation of the radioactive sugars from the uronie acids by means of a Dowex I acetate column. Paper chromatography in ethyl a c e t a t e - p y r i d i n e water (2 : I : I) (Solvent I) and n - b u t a n o l - p y l i d i n e - w a t e r - a c e t i c acid (60:40:3 ° : 3) (Solvent II) showed that the hydrolysate contained predominately xylose, some arabinose and several unidentified substances. The uronic acids from hemicellulose B were separated in ethyl acetate-acetic acid-formic acid-water (18:3:i:4) (Solvent III). Fig. I shows that most of the radioactivity was located in the two aldobiouronic acids glucurono-galactose and glucurono-xylose. Small amounts of galacturonic acid and glucuronic acid (separated in Solvent I), were also present. However, the amount of glucuronic acid was low, as after the hydrolysis it passed through the Dowex I column as glucuronolactone and was thus included in the sugar fraction. The two aldobiouronic acids were further identified by chromatography in p h e n o l - w a t e r formic acid (IOO: IOO: I, w/v/v) (Solvent IV) and Solvent I. On hydrolysis with I M HC1 for 4 h and chromatography in Solvents II and III they gave spots of glucuronic acid and glucuronolactone. Reduction with NaBH 4 of the carboxyl group of glucuronogalaetose followed by acid hydrolysis resulted in the formation of labelled glucose, which was identified in Solvents I and II.
•~4oc
~
£~200 o. '~
~00 '
U
Sto~rt
1 ~ GIC UA'Gol
,
,
,
20 i i GIc UA-Xyl
J
i
J
30 40 ~ i No.of QScrn GolUA ,GicUA str'ip
Fig. i. Paper chromatographic pattern (Solvent II[) of the radioactivity of the uronic acid mixture resulting from acid hydrolysis of the hemicellulose 13 fraction formed from UDP-[14C]glucuronic acid. Material corresponding to experiment " + UDP-[I~C 1xylose" from Table I. Minor peaks representing glucuronolactone and other fast-moving unidentified substances are not shown.
According to the known chromatographic data 7 and the results of the degradation experiments the identity of the two aldobiouronic acids is well established. Their occurrence proves that the newly introduced glucuronic acid was covalently linked to polysaccharides containing galactose and xylose, most likely glucuronogalactans and glucurono-xylans. The acceptor molecules for the glucuronic acid seem to be present in the particulate enzyme preparation; a similar situation was found in the studies on the introduction of methyl ether groups into hemicellulose B (ref. 5) and methyl ester groups into pectin 8. This at least seems to be the case for the Biochim. Biophys. Acta, 148 (1967) 572-574
574
SHORT COMMUNICATIONS
glucurono-galactan. No nucleoside diphosphate galactose was present in the incubation mixture which could have made it possible that the glucuronic acid molecule was introduced to the growing end of a galactan chain or was first combined with galactose and then transferred as a preformed unit to polysaccharide-like acceptors. The suggestion that the glucuronic acid residue is introduced into preformed polysaccharides has to be studied in more detail. It has the remarkable consequence that the mode of biosynthesis of plant cell wall heteropolysaccharides is quite different from that of several bacterial cell-wall heteropolysaccharides, which are built from preformed units with a definite monomer sequence 9. The particulate enzyme preparation obviously contains, besides the transferase for glucuronic acid, a number of other enzymes necessary for the formation of the cell wall. The UDP-glucuronic acid seems to be epimerized to UDP-galacturonic acid, decarboxylated to UDP-xylose and the latter epimerized to UDP-arabinose. In addition, the sugar parts of the different nucleotides formed are introduced into polysaccharides. This is indicated by tile products synthesized and also illustrated by the experiment where UDP-[12C~xylose was added (Table I). In this experiment the radioactive UDP-xylose formed by decarboxylation was diluted b y tile non-radioactive nucleotide which resulted in less incorporation of labelled sugars into polysaccharides. Most of the enzymes responsible for the observed interconversions of nucleotide sugars and the transfer of their sugar residues have already been demonstrated 1°. Their occurrence together with acceptor polysaccharides in the particulate enzyme preparation made a detailed study of the properties of the glucuronic acid-transferring enzyme impossible. On the other hand, it makes the ultrastructural organisation and the cytological origin of the cell-wall-forming particulate material a very attractive study. Part of this work was done during a stay at the Department of Biochemistry, University of California, Berkeley, Calif. I wish to thank Dr. W. Z. HASSlD for his generous hospitality and Dr. R. W. BAILEY for m a n y helpful suggestions.
Imtitut fiir Angewa~dte Bota~zik der Technischen Hochschule Miinche~, Miinche~ (Germa~,y) I 2 3 4 5 6 7 8 9 io
H. KAuss
T. E. TIMELL, Advan. Carbohydrate Chem., 19 (1964) 247. T. E . TIMELL, Advan. Carbohydrate Chem., 20 (1965) 409. G. O. ASPINALL, Advan. Carbohydrate Chem., 14 (1959) 429 • B. D. E. GAILLARD,Phytochemislry, 4 (1965) 631. H . KAUSS AND W . Z. HASSlD, J. Biol. Chem., 242 (1967) 168o. E. O. CASTANERA AND W . Z. HASSlD, Arch. Biochem. Biophys., I i O (1965) 462. P. M. RAY AND D. A. I~OTTENBERG,Biochem. J., 90 (1964) 646. H. K A u s s , A. L. SWANSON AND V~r. Z. HASSID, Biochem. Biophys. Res, Commun., 26 (1967) 234. B. L. HORECKER, A n n . Rev. 2VIicrobiol., 20 (1966) 253. V~T. Z. HASSlD, Ann. Rev. Plant Physiol., 18 (1967) 253.
Received August I6th, 1967 Biochim. Biophys. Acta, 148 (1967) 572-574
SHORT
COMMUNICATIONS
BBA 2337 I
Biosynthesis of the glucuronic acid unit of hemicellulose B from UDP-glucuronic acid The term hemicellulose designates a chemically ill-defined group of plant cellwall heteropolysaccharides, which may be composed of different monosaccharides with various types of linkages. A feature in common seems to be the occurrence of single molecule side chains of glucuronic acid or 4-0-methylglucuronic acid, the proportion of the uronic acid being higher in the hemicellulose B fraction than in the hemicellulose A fraction I 4. It was recently shown for the 4-0-methyl-glucurono-xylan from corn cobs that the 4-0-methyl ether group is introduced into preformed polysaccharide molecules s. Experiments on the biosynthesis of the glucuronic acid unit of hemicellulose B are reported here. They indicate that the particulate enzyme preparation from immature corn cobs contains, in addition to the methyl transferase, an enzyme which introduces the glucuronic acid group from UDP-glucuronic acid into hemicellulose B. All materials, operations and methods for the analysis of the polysaccharides are essentially the same as in the study on the formation of the methyl ether s . Labelled UDP-glucuronic acid was prepared by enzymatic dehydrogenation of UDP-glucose a. 6-/3-Glucuronosyl-galactose was a gift from Dr. G. O. ASPINALL. TABLE I LABELLED PRODUCTS FORMED FROM
UDP-I14C]GLUCURONIC
RATIONS
ABSENCE
FROM
CORN
COBS IN
THE
AND
ACID BY PARTICULATE ENZYME PREPA-
PRESENCE
OF
UDp-F12C]XYLOSF.
The incubation m i x t u r e s contained 2oo/~1 enzyme preparation, 2o0 ill p h o s p h a t e buffer (o.i M, p H 6.3), i ~o albumin, o. 4 M sucrose, IOO m/,moles MgC12, 6.8 nil, moles (696ooo counts/mini UDP-I14C~]glucuronic acid and no, or 12o ml, moles U D P - I I ~ Q x y l o s e (final p H 6.5). The m i x t u r e was incubated at 3 °0 for 15 min and the reaction stopped by addition of 2 ml boiling 99 ~o ethanol. The material was washed twice with 2 ml 75 % ethanol, extracted twice with i ml of o. 5 % amm o n i u m oxalate a n d t h e n with 2 ml of 2 ~o N a O H , each step with heating to 80 ° for 5 min. The p o l y s a c c h a r i d e s were precipitated from the a m m o n i u m oxalate solution by addition of 5 vol. 99 ~o ethanol. The N a O H solution was b r o u g h t to p H 4.7 with acetic acid, the precipitate (hemicellulose A) collected and t h e n t h e h e m i c e l l u l o s e B fraction precipitated by addition of 5 vol. 99 °/o ethanol.
Incorporation inlo polysaccharides
. UDP-[aeCqXyl (counts/rain "z ±o a)
+ UDP-[Iz2QA'y! (counts/rain >4 ±o a)
Total Soluble in a m m o n i u m oxalate (total) Precipitated from a n l m o n i u m oxalate Acid hydrolysate of ppt. from a m m o n i u n l oxalate: neutral sugars uronic acids Soluble in N a O H (total) : helnicellulose A hemicellulose B Acid hydrolysis of h e m i e e l l u l o s e B: n e u t r a l sugars uronic acids
395 23o I76"
03 35 22
129. 4 2. 3 165 62 IOO
12.6 3.8 29 i2 15
76.8 4. i
7.1 4.8
* 9o °
Biochim. Biophys. Acta, I,i8 (1967) 572-574
SHORT COMMUNICATIONS
573
On incubation of the particulate enzyme preparation with UDP-E14Ce]glucuronic acid, more than half of the radioactivity was incorporated into polysaccharides (Table I). Fractional elution and precipitation, together with acid hydrolysis and paper chromatography, were used to characterize these products. An appreciable part of the material was solubilized with a solution of ammonium oxalate. Although the proportion of uronic acids in this fraction was smaller than in the hemicellulose B fraction, essentially the same radioactive sugars and uronic acids as in hemicellulose B were present. The hemicellulose B fraction was characterized in greater detail by acid hydrolysis and separation of the radioactive sugars from the uronie acids by means of a Dowex I acetate column. Paper chromatography in ethyl a c e t a t e - p y r i d i n e water (2 : I : I) (Solvent I) and n - b u t a n o l - p y l i d i n e - w a t e r - a c e t i c acid (60:40:3 ° : 3) (Solvent II) showed that the hydrolysate contained predominately xylose, some arabinose and several unidentified substances. The uronic acids from hemicellulose B were separated in ethyl acetate-acetic acid-formic acid-water (18:3:i:4) (Solvent III). Fig. I shows that most of the radioactivity was located in the two aldobiouronic acids glucurono-galactose and glucurono-xylose. Small amounts of galacturonic acid and glucuronic acid (separated in Solvent I), were also present. However, the amount of glucuronic acid was low, as after the hydrolysis it passed through the Dowex I column as glucuronolactone and was thus included in the sugar fraction. The two aldobiouronic acids were further identified by chromatography in p h e n o l - w a t e r formic acid (IOO: IOO: I, w/v/v) (Solvent IV) and Solvent I. On hydrolysis with I M HC1 for 4 h and chromatography in Solvents II and III they gave spots of glucuronic acid and glucuronolactone. Reduction with NaBH 4 of the carboxyl group of glucuronogalaetose followed by acid hydrolysis resulted in the formation of labelled glucose, which was identified in Solvents I and II.
•~4oc
~
£~200 o. '~
~00 '
U
Sto~rt
1 ~ GIC UA'Gol
,
,
,
20 i i GIc UA-Xyl
J
i
J
30 40 ~ i No.of QScrn GolUA ,GicUA str'ip
Fig. i. Paper chromatographic pattern (Solvent II[) of the radioactivity of the uronic acid mixture resulting from acid hydrolysis of the hemicellulose 13 fraction formed from UDP-[14C]glucuronic acid. Material corresponding to experiment " + UDP-[I~C 1xylose" from Table I. Minor peaks representing glucuronolactone and other fast-moving unidentified substances are not shown.
According to the known chromatographic data 7 and the results of the degradation experiments the identity of the two aldobiouronic acids is well established. Their occurrence proves that the newly introduced glucuronic acid was covalently linked to polysaccharides containing galactose and xylose, most likely glucuronogalactans and glucurono-xylans. The acceptor molecules for the glucuronic acid seem to be present in the particulate enzyme preparation; a similar situation was found in the studies on the introduction of methyl ether groups into hemicellulose B (ref. 5) and methyl ester groups into pectin 8. This at least seems to be the case for the Biochim. Biophys. Acta, 148 (1967) 572-574
574
SHORT COMMUNICATIONS
glucurono-galactan. No nucleoside diphosphate galactose was present in the incubation mixture which could have made it possible that the glucuronic acid molecule was introduced to the growing end of a galactan chain or was first combined with galactose and then transferred as a preformed unit to polysaccharide-like acceptors. The suggestion that the glucuronic acid residue is introduced into preformed polysaccharides has to be studied in more detail. It has the remarkable consequence that the mode of biosynthesis of plant cell wall heteropolysaccharides is quite different from that of several bacterial cell-wall heteropolysaccharides, which are built from preformed units with a definite monomer sequence 9. The particulate enzyme preparation obviously contains, besides the transferase for glucuronic acid, a number of other enzymes necessary for the formation of the cell wall. The UDP-glucuronic acid seems to be epimerized to UDP-galacturonic acid, decarboxylated to UDP-xylose and the latter epimerized to UDP-arabinose. In addition, the sugar parts of the different nucleotides formed are introduced into polysaccharides. This is indicated by tile products synthesized and also illustrated by the experiment where UDP-[12C~xylose was added (Table I). In this experiment the radioactive UDP-xylose formed by decarboxylation was diluted b y tile non-radioactive nucleotide which resulted in less incorporation of labelled sugars into polysaccharides. Most of the enzymes responsible for the observed interconversions of nucleotide sugars and the transfer of their sugar residues have already been demonstrated 1°. Their occurrence together with acceptor polysaccharides in the particulate enzyme preparation made a detailed study of the properties of the glucuronic acid-transferring enzyme impossible. On the other hand, it makes the ultrastructural organisation and the cytological origin of the cell-wall-forming particulate material a very attractive study. Part of this work was done during a stay at the Department of Biochemistry, University of California, Berkeley, Calif. I wish to thank Dr. W. Z. HASSlD for his generous hospitality and Dr. R. W. BAILEY for m a n y helpful suggestions.
Imtitut fiir Angewa~dte Bota~zik der Technischen Hochschule Miinche~, Miinche~ (Germa~,y) I 2 3 4 5 6 7 8 9 io
H. KAuss
T. E. TIMELL, Advan. Carbohydrate Chem., 19 (1964) 247. T. E . TIMELL, Advan. Carbohydrate Chem., 20 (1965) 409. G. O. ASPINALL, Advan. Carbohydrate Chem., 14 (1959) 429 • B. D. E. GAILLARD,Phytochemislry, 4 (1965) 631. H . KAUSS AND W . Z. HASSlD, J. Biol. Chem., 242 (1967) 168o. E. O. CASTANERA AND W . Z. HASSlD, Arch. Biochem. Biophys., I i O (1965) 462. P. M. RAY AND D. A. I~OTTENBERG,Biochem. J., 90 (1964) 646. H. K A u s s , A. L. SWANSON AND V~r. Z. HASSID, Biochem. Biophys. Res, Commun., 26 (1967) 234. B. L. HORECKER, A n n . Rev. 2VIicrobiol., 20 (1966) 253. V~T. Z. HASSlD, Ann. Rev. Plant Physiol., 18 (1967) 253.
Received August I6th, 1967 Biochim. Biophys. Acta, 148 (1967) 572-574