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Biosynthesis of alginate. Epimerisation of d -mannuronic to l -guluronic acid residues in the polymer chain
BIOCHIMICA
ET BIOPHYSICA
PRELIMINARY
557
ACTA
NOTES
BRA 21273
Biosynthesis of alginate. Epimerisation
of D-mannuronic
to L-guluronic acid
residues in the polymer chain Two different
mechanisms
are recognised
for the biosynthesis
of heteropoly-
saccharides. In chondroitin sulphate l, for example, there is a stepwise addition of monosaccharide residues to the nonreducing end of a growing chain. N-Acetylgalactosamine is added only to oligosaccharides with nonreducing terminal uranic acid residues, while glucuronic acid is added only to oligosaccharides with N-acetylgalactosamine as nonreducing terminal residues, thus leading to an alternating structure. On the other hand, in the biosynthesis of O-antigen of Salmonella newington, lipid-bound trisaccharides are first synthesised and then transferred to the reducing end of a growing polysaccharide chairP. We now present evidence for another type of heteropolysaccharide synthesis, in which one of the monomers is transformed into its C(5) epimer on the polymer level with very little concomitant depolymerisation. The heteropolysaccharide we have investigated is alginate, a linear polyuronide composed of the C(5) epimeric monomers D-mannuronic and L-guluronic acid in varying proportions. The two monomers are distributed in a block-wise fashion, long groups of contiguous n-mannuronic acid residues and similar groups of L-guluronic acid residues being joined through regions containing a predominantly alternating sequence of the same monomers3-6. Alginate is a constituent of all brown algae and has recently, in a partly acetylated form, also been found as an extracellular product of Azotobacter vinelamW and certain strains of Pseudomonas aeruginosa7~s. In a study of the production of alginate by a strain by Kjell Eimhjellen, Institute of Technical Biochemistry,
of A. vinelandii (isolated N.T.H., Trondheim), we
observed that the relative amounts of the two monomers (mannuronic acid/guluronic acid ratiog) in the alginate produced depended upon the amount of Ca2+ present in the nutrient. Moreover, if Ca2+ was added during the production of alginate, a change took place in the composition of the alginate already synthesised. This was confirmed by the following experiment: A culture of A. v&elan&i, with a nutrient containing 0.1 n0I CaCl,, was harvested after 72 h. The supernatant, after removal of the cells by centrifugation, was divided into two parts; both of which were incubated at 30’ for 20 h, one with the Ca2+ concentration increased to 3.4 mM, the other without any addition of CaCl,. Toluene was added to prevent bacterial growth. The mannuronic acid/guluronic acid ratio of the isolated samples of alginate was 3.75 (79 y0 mannuronic acid) without addition of CaCl,, and 1.5 (60% mannuronic acid) after incubation in the presence of an increased amount of Ca2+. The results suggested that the supernatant contained an enzyme capable of epimerising residues of mannuronic acid to residues of guluronic acid in the polymer B&him.
Biophys.
Acta,
192 (1969) 557-55g
558
PRELIMINARY
NOTES
chain, depending upon the amount of Ca2+ present in the solution. Further evidence for the presence of such an enzyme was obtained as follows: A liquid culture of A. vinelandii (ZOOml), grown in the presence of 0.1 mM CaCl,, was centrifuged after 48 h, and (NHJ2S0, was added to the supernatant at o” to 80 y0 saturation. The mixture was centrifuged and the precipitate dissolved in and dialysed against a solution containing the same amounts of inorganic constituents as the nutrient, but with the CaCl, omitted. The preparation (20 ml) contained 8.6 mg carbohydrate, as measured by the phenol-sulphuric acid reactionll (expressed as guluronic acid) and 24.7 mg protein, measured by the Folin-Ciocalteu method, with bovine serum albumin as the arbitrary standard. It was mixed with an aqueous solution (40 ml) containing IOO mg alginate (10% moisture,
I
FRhCTIONATION OF PARTIALLY
DEGRADED
,Wannuronic acid (%)
ALGINATE
Hydrolysis, Soluble
BEFORE
Biochim.
92
9.1 jI.0
49
Biophys.
Acta,
192 (1969)
557-559
AFTER
2 h, 0.3 Il4 HCI,
ENZYME
IOOO (%)
Insoluble Soluble, pH 2.85
Before After
AND
Precipitated, pH 2.85
80.5
10.4
IF.2
51.8
TREATMENT
PRELIMINARY
NOTES
559
Fig. I compares the results obtained in this way with the original and enzymemodified alginate, respectively. These results indicate that the enzyme-modified alginate has a block structure similar to that of ordinary algal alginate of the same uranic acid composition57 15.
Fig. I. Ascending boundaries (IO mA, 30 min, 0.05 M NaCl + 0.75 mM CaCl,) of the insoluble fraction after 2 h of hydrolysis, 100’ in 0.3 M HCl. Upper: before enzyme treatment; lower: after enzyme treatment.
Whether the synthesis of alginates in brown algae takes place by modification of previously synthesised polymannuronic acid through the action of an enzyme similar to that described above from A. vinelandii remains to be established. Observations reported by HELLEBUST AND HAUG’~ appear, however, significant in this connection. By following the incorporation of 14C into alginate in Laminaria digitata plants, and fractionating the alginate after hydrolysis as described above, they found that in light, where the synthesis of alginate was rapid, the activity was mainly incorporated into the fragments rich in mannuronic acid. When the plants were subsequently kept in darkness, the increase of activity in the alginate fraction was small, but the activity became more evenly distributed between the different alginate fragments. Norwegian Trondheim
I 2 3 4 5
6 7 8 9 IO II
12 13 14 15 16
ARNE
Institute of Seaweed Research, N.T.H., (Norway)
HAUG
B J~RN LARSEN
h. P. ri. A. A.
TELSER, H. C. ROBINSON AND A. DORFMAN, Arch. Biochem. Biophys., 116 (1966) 458. W. ROBBINS, P. BRAY, M. DANKERT AND A. WRIGHT, Science, 158 (1967) 1536. HAUG, B. LARSEN AND 0. SII~IDSRQD, Acta Chem. &and., 20 (1966) 183. HAUG, B. LARSEN AND 0. SMIDSR~D, Acta Chem. Stand., 21 (1967) 691. HAUG, S. MYKLESTAD, B. LARSEN AND 0. SMIDSROD, Acta Chem. &and., 21 (1967) 768. P. r\. J. GORIN AND J. F. T. SPENCER, Can. J. Chem., 44 (1966) 993. -1. LINKER AND R. S. JONES, J. Biol. Chem., 241 (1966) 3845. D. M. CARLSON AND L. R. MATTHEWS, Biochemistry, 5 (1966) 2817. X. HAUG AND B. LARSEN, Acta Chem. &and., 16 (1962) 1908. J. R. NORRIS AND H. L. JENSEN, Arch. Mikrobiol., 31 (1958) 198. 11. DUBOIS, K. A. GILLES, J. K. HAMILTON, P. H. REBERS AND F. SMITH, Anal. Chem.,
(1956) 350. J. E. SCOTT, Methods Biochem. Anal., 8 (1960) 163. A. HAUG AND 0. SMIDSRBD, Acta Chem. &and., 16 (1962) 1569. 0. SMIDSROD AND A. HAUG, Acta Chem. &and., 22 (1968) 797. -1. HAUG AND B. LARSEN, Proc. 6th Intern. Seaweed Symp., Santiago, Spain, published. J. HELLEBUST AND A. HAUG, Proc. 6th Intern. Seaweed Symp., Santiago, Spain, published.
Received
August
rdth,
1968,
28
to be
1968, to be
1969 Biochim.
Biophys.
Acta,
Igz (1969) 557-559
ET BIOPHYSICA
PRELIMINARY
557
ACTA
NOTES
BRA 21273
Biosynthesis of alginate. Epimerisation
of D-mannuronic
to L-guluronic acid
residues in the polymer chain Two different
mechanisms
are recognised
for the biosynthesis
of heteropoly-
saccharides. In chondroitin sulphate l, for example, there is a stepwise addition of monosaccharide residues to the nonreducing end of a growing chain. N-Acetylgalactosamine is added only to oligosaccharides with nonreducing terminal uranic acid residues, while glucuronic acid is added only to oligosaccharides with N-acetylgalactosamine as nonreducing terminal residues, thus leading to an alternating structure. On the other hand, in the biosynthesis of O-antigen of Salmonella newington, lipid-bound trisaccharides are first synthesised and then transferred to the reducing end of a growing polysaccharide chairP. We now present evidence for another type of heteropolysaccharide synthesis, in which one of the monomers is transformed into its C(5) epimer on the polymer level with very little concomitant depolymerisation. The heteropolysaccharide we have investigated is alginate, a linear polyuronide composed of the C(5) epimeric monomers D-mannuronic and L-guluronic acid in varying proportions. The two monomers are distributed in a block-wise fashion, long groups of contiguous n-mannuronic acid residues and similar groups of L-guluronic acid residues being joined through regions containing a predominantly alternating sequence of the same monomers3-6. Alginate is a constituent of all brown algae and has recently, in a partly acetylated form, also been found as an extracellular product of Azotobacter vinelamW and certain strains of Pseudomonas aeruginosa7~s. In a study of the production of alginate by a strain by Kjell Eimhjellen, Institute of Technical Biochemistry,
of A. vinelandii (isolated N.T.H., Trondheim), we
observed that the relative amounts of the two monomers (mannuronic acid/guluronic acid ratiog) in the alginate produced depended upon the amount of Ca2+ present in the nutrient. Moreover, if Ca2+ was added during the production of alginate, a change took place in the composition of the alginate already synthesised. This was confirmed by the following experiment: A culture of A. v&elan&i, with a nutrient containing 0.1 n0I CaCl,, was harvested after 72 h. The supernatant, after removal of the cells by centrifugation, was divided into two parts; both of which were incubated at 30’ for 20 h, one with the Ca2+ concentration increased to 3.4 mM, the other without any addition of CaCl,. Toluene was added to prevent bacterial growth. The mannuronic acid/guluronic acid ratio of the isolated samples of alginate was 3.75 (79 y0 mannuronic acid) without addition of CaCl,, and 1.5 (60% mannuronic acid) after incubation in the presence of an increased amount of Ca2+. The results suggested that the supernatant contained an enzyme capable of epimerising residues of mannuronic acid to residues of guluronic acid in the polymer B&him.
Biophys.
Acta,
192 (1969) 557-55g
558
PRELIMINARY
NOTES
chain, depending upon the amount of Ca2+ present in the solution. Further evidence for the presence of such an enzyme was obtained as follows: A liquid culture of A. vinelandii (ZOOml), grown in the presence of 0.1 mM CaCl,, was centrifuged after 48 h, and (NHJ2S0, was added to the supernatant at o” to 80 y0 saturation. The mixture was centrifuged and the precipitate dissolved in and dialysed against a solution containing the same amounts of inorganic constituents as the nutrient, but with the CaCl, omitted. The preparation (20 ml) contained 8.6 mg carbohydrate, as measured by the phenol-sulphuric acid reactionll (expressed as guluronic acid) and 24.7 mg protein, measured by the Folin-Ciocalteu method, with bovine serum albumin as the arbitrary standard. It was mixed with an aqueous solution (40 ml) containing IOO mg alginate (10% moisture,
I
FRhCTIONATION OF PARTIALLY
DEGRADED
,Wannuronic acid (%)
ALGINATE
Hydrolysis, Soluble
BEFORE
Biochim.
92
9.1 jI.0
49
Biophys.
Acta,
192 (1969)
557-559
AFTER
2 h, 0.3 Il4 HCI,
ENZYME
IOOO (%)
Insoluble Soluble, pH 2.85
Before After
AND
Precipitated, pH 2.85
80.5
10.4
IF.2
51.8
TREATMENT
PRELIMINARY
NOTES
559
Fig. I compares the results obtained in this way with the original and enzymemodified alginate, respectively. These results indicate that the enzyme-modified alginate has a block structure similar to that of ordinary algal alginate of the same uranic acid composition57 15.
Fig. I. Ascending boundaries (IO mA, 30 min, 0.05 M NaCl + 0.75 mM CaCl,) of the insoluble fraction after 2 h of hydrolysis, 100’ in 0.3 M HCl. Upper: before enzyme treatment; lower: after enzyme treatment.
Whether the synthesis of alginates in brown algae takes place by modification of previously synthesised polymannuronic acid through the action of an enzyme similar to that described above from A. vinelandii remains to be established. Observations reported by HELLEBUST AND HAUG’~ appear, however, significant in this connection. By following the incorporation of 14C into alginate in Laminaria digitata plants, and fractionating the alginate after hydrolysis as described above, they found that in light, where the synthesis of alginate was rapid, the activity was mainly incorporated into the fragments rich in mannuronic acid. When the plants were subsequently kept in darkness, the increase of activity in the alginate fraction was small, but the activity became more evenly distributed between the different alginate fragments. Norwegian Trondheim
I 2 3 4 5
6 7 8 9 IO II
12 13 14 15 16
ARNE
Institute of Seaweed Research, N.T.H., (Norway)
HAUG
B J~RN LARSEN
h. P. ri. A. A.
TELSER, H. C. ROBINSON AND A. DORFMAN, Arch. Biochem. Biophys., 116 (1966) 458. W. ROBBINS, P. BRAY, M. DANKERT AND A. WRIGHT, Science, 158 (1967) 1536. HAUG, B. LARSEN AND 0. SII~IDSRQD, Acta Chem. &and., 20 (1966) 183. HAUG, B. LARSEN AND 0. SMIDSR~D, Acta Chem. Stand., 21 (1967) 691. HAUG, S. MYKLESTAD, B. LARSEN AND 0. SMIDSROD, Acta Chem. &and., 21 (1967) 768. P. r\. J. GORIN AND J. F. T. SPENCER, Can. J. Chem., 44 (1966) 993. -1. LINKER AND R. S. JONES, J. Biol. Chem., 241 (1966) 3845. D. M. CARLSON AND L. R. MATTHEWS, Biochemistry, 5 (1966) 2817. X. HAUG AND B. LARSEN, Acta Chem. &and., 16 (1962) 1908. J. R. NORRIS AND H. L. JENSEN, Arch. Mikrobiol., 31 (1958) 198. 11. DUBOIS, K. A. GILLES, J. K. HAMILTON, P. H. REBERS AND F. SMITH, Anal. Chem.,
(1956) 350. J. E. SCOTT, Methods Biochem. Anal., 8 (1960) 163. A. HAUG AND 0. SMIDSRBD, Acta Chem. &and., 16 (1962) 1569. 0. SMIDSROD AND A. HAUG, Acta Chem. &and., 22 (1968) 797. -1. HAUG AND B. LARSEN, Proc. 6th Intern. Seaweed Symp., Santiago, Spain, published. J. HELLEBUST AND A. HAUG, Proc. 6th Intern. Seaweed Symp., Santiago, Spain, published.
Received
August
rdth,
1968,
28
to be
1968, to be
1969 Biochim.
Biophys.
Acta,
Igz (1969) 557-559