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Polysaccharide Biosynthesis
P. W. ROBBINS, A. WRIGHT, and M. DANKERT From the Division of Biochemistry, Massachusetts Institute of Technology, Cambridge
AB ST R ACT Polysaccharide synthesis is discussed from the point of view of the sources of biological information that determine the structures and control the rates of synthesis of complex polysaccharides. It is concluded that three types of information contribute in important and different ways, namely enzyme specificity, primer substances, and the structure of the cytoplasmic membrane. Each of these factors is discussed in a general way with examples of its contri- bution to the structure and organization of specific polysaccharides.
The general problem of polysaccharide synthesis may be considered from a number of points of view. It is possible, for instance, to divide polysaccharides into groups on the basis of chemical complexity and^ it^ would be^ feasible^ to discuss homopolysaccharides, polysaccharides with simple repeating units, and complex heteropolysaccharides as three distinct problems of biosynthesis. Alternatively, it would be possible to separate polysaccharides into functional groups and consider as separate topics the synthesis of intracellular storage polysaccharides, cell wall structural polysaccharides, and extracellular or capsular polysaccharides. Both approaches are reasonable and it is possible to develop the themes that the biochemical mechanisms of biosynthesis be- come more complex as one considers polysaccharides with increasing struc- tural complexity or increasing functional complexity. It is obvious that more enzymes will be involved in the formation of the variety of linkages of a complex heteropolysaccharide than will be required to form the one or two types of linkage in a homopolysaccharide. It is also obvious that a complex function may require the intervention of enzymes, cofactors, and control systems to ensure the coordination of polysaccharide synthesis with the forma- tion of other substances, as in the synthesis of the bacterial cell wall, or to regulate synthesis and degradation, as in the case of glycogen formation and breakdown. A third way of discussing polysaccharide synthesis is to consider the sources of biological information that determine the structure and control the syn- thesis of polysaccharides. This is the point of view that will be taken in the present paper and it will be assumed that there are primarily three sources of information, namely enzyme specificity, primer substances, and the struc- ture of the cytoplasmic membrane. These sources of information may well be
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The Journal of General Physiology
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APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES
related to one another. At the present stage of understanding in molecular
biology, however, each of these components may be considered as an inde-
pendent source of information and, in each case, the source of a different
type of information. Enzyme specificity derives from protein structure (ulti-
mately from nucleic acid nucleotide sequences) and specific enzymes are
responsible for the synthesis of activated monosaccharides, the nucleoside
diphosphate sugars, and for the specific transfer of glycosyl residues from these
nucleoside diphosphate sugars to acceptor molecules,^ usually^ other^ saccharide
residues. Primers may be defined as^ substances^ that^ cannot^ be^ derived^ readily
de novo from enzymes and metabolites; they serve as enzyme substrates^ and
probably also direct enzyme action by^ allosteric^ interactions^ and^ structural
effects. The cytoplasmic membrane has recently come to^ the foreground^ as
the site of cell wall polysaccharide synthesis and, as^ will^ be^ mentioned^ below,^ a
phospholipid that is probably present^ in^ the^ cytoplasmic^ membrane^ serves
as a coeznyme in the synthesis of at least some cell wall polysaccharides.
What determines the structure of the cytoplasmic membrane is at present
almost purely a matter of speculation and, in fact, Sonneborn (1) has pointed
out that the relationship between the cell genome and the structure and func-
tion of the cytoplasmic membrane remains one of the most important un-
explored areas of biology. To summarize, therefore, enzyme specificity,
primer materials, and the structure of the cytoplasmic membrane may be
considered as the three basic sources that determine and control the syn-
thesis of polysaccharides, although all three factors may not operate directly
in every case.
The possibility that other sources of information may function in poly-
saccharide synthesis has been proposed. McMullen (2) has suggested that
"nucleoside diphosphate saccharides... are analogous to the activated
amino acid in^ protein^ template^ biogenesis^ and^ correspondingly^ enter^ into
the same kind of ... coding mechanisms involving polynucleotide struc-
tures .... " Such speculation is interesting but, as far as we are aware, has
no experimental basis.
Enzyme Specificity
Little comment is required on the major role played by enzyme specificity
in determining and controlling polysaccharide synthesis. The synthesis of
most polysaccharides begins with the formation of a nucleoside diphosphate
glycosyl derivative from a nucleotide triphosphate and a glycosyl phosphate
ester. Fig. 1 illustrates the formation of uridine diphosphate glucose (^ =^ UDP-
glucose) from^ uridine^ triphosphate^ and^ glucose-l-phosphate.^ Even^ at^ this
initial stage the reaction shows a high degree of specificity and control.
Beinstein and Robbins (3), for example, studied the formation of the struc-
turally similar nucleotides UDP-glucose and thymidine diphosphate glucose
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APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES
( =TDP-glucose) (^) by extracts of enteric bacteria. It was shown that the nucleo- tides are formed by separate (^) soluble enzymes, each with its own pattern of feed-back control. (^) Approximately sixty nucleoside diphosphate sugars are known at the present (^) time. Some of these are formed directly from an a-D- glycosyl-l-phosphate (^) and a nucleoside triphosphate, but many are products of oxidation, reduction, and epimerization (^) reactions that are catalyzed by specific soluble enzymes. For example, (^) such reactions bring about the trans- formation of TDP-glucose (^) to TDP-rhamnose, the direct precursor of poly- saccharide rhamnose. The known (^) systems are summarized in the reviews by Leloir (4) and Ginsburg (^) (5) and will not be enumerated here. The (^) enzymes that catalyze the transfer of glycosyl residues from nucleoside diphosphate (^) sugars to the oxygen residues of other saccharide moieties in general show (^) absolute specificity with respect to the linkage position and anomeric configuration. (^) Since, as far as is known, all nucleoside diphosphate sugars (^) have the same absolute configuration (=a-D) at the phosphate- glycosyl linkage (^) some transfer reactions clearly take place with retention of this (^) configuration at the glycosyl position while other transfer reactions lead to inversion of configuration. In either case the energy of the system is pre- served, presumably by way of covalent intermediates; thus, it is possible (^) that transfer (^) reactions that take place with retention of the glycosyl carbon atom configuration are actually double inversion reactions. According (^) to this concept (^) (6) the enzyme that will catalyze transfer with retention of configura- tion reacts with the nucleoside diphosphate sugar with displacement of the nucleoside diphosphate and the formation of an enzyme-sugar covalent intermediate. This intermediate has the inverted glycosyl configuration (^) and reaction with the final acceptor in a second displacement reaction leads to restoration of the original glycosidic configuration. Reactions that involve over-all inversion, on the other hand, would be visualized (^) as simple dis- placement reactions on the nucleoside diphosphate sugar glycosyl (^) linkage by the acceptor group oxygen moiety. While these (^) simple schemes would account adequately for the occurrence of both types of glycosidic linkages (^) in polysaccharides, little serious work hasyet been carried out on the mechanisms of transfer reactions.
The Role of Primer in PolysaccharideBiosynthesis
Although enzymatic (^) specificity adequately accounts for the sequential ad- dition of subunits into correct linkage (^) with growing polysaccharide chains, it appears that the entire assembly of the molecule (^) cannot be achieved by enzymes, coenzymes, and metabolites alone. (^) An interesting example of such an instance is the biosynthesis of glycogen. (^) Glycogen is composed of several hundred to several thousand chains of a-1 ,4-linked D-glucose units, the interchain linkage being of the a-1,6-glucosidic type (Fig. 2). It is synthe-
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P. W. ROBBINS, A. WRIGHT, AND M. DANKERT Polysaccharide Biosynthesis 335
sized by the transfer of glucose units from UDP-glucose to the nonreducing end groups of primer glycogen, catalyzed by the enzyme glycogen synthetase (7). The action of the enzyme is to lengthen the exterior chains of the molecule (Fig. 3). Branching of these newly created chains is catalyzed by amylo- 1,4 - 1,6-transglucosidase (branching enzyme), the over-all effect of the two enzymes being to cause the outward growth of the molecule. Glycogen itself is the best acceptor or primer in the glycogen synthetase reaction, al- though oligosaccharides as small as maltose may also act as acceptors, but at a very much reduced rate (8). The question of how the first glycogen molecules appear in the cell raises two alternatives. Either glycogen is synthesized de novo from precursors present in the cell or preformed glycogen is present at the birth of each new cell. De novo synthesis by glycogen synthetase does not seem likely at present since
6 G G ~G (^) G ,G G G GG +UDP UDPG' + 'IN FIGURE 3. Action of glycogen synthetase. The figure shows transfer of glucose from UDP-glucose to the nonreducing end group of a glycogen molecule resulting in chain elongation.
glucose and other common metabolites do not act as acceptors in the reaction. As mentioned previously, this enzyme is able to use maltose as an acceptor at a slow rate, but since maltose and its homologues were shown by Ola- varria (9) to be degradation products of glycogen rather than its precursors, this pathway seems unlikely. Brown (10), in fact, has reported that no de novo glycogen synthesis can be detected when purified glycogen synthetase is incubated with UDP-glucose in the absence of primer glycogen. The de novo synthesis of glycogen from glucose-l-phosphate by a mixture of highly purified, primer-free phosphorylase and branching enzyme has been reported by Brown et al. (11). It is suggested that the process involves the formation of a glucosyl enzyme which becomes the acceptor for chain growth. However, such a reaction in vivo would require a favorable glucose- - phosphate to inorganic phosphate ratio which would necessitate compart- mentalization of the cell, since this ratio normally favors the phosphorolysis reaction. It has also been observed that the appearance of glycogen in chick embryo precedes by a significant period the appearance of phosphorylase (12). Furthermore, it has been shown that in McArdle's disease, which is characterized by an accumulation of glycogen in muscle, there is a complete absence of phosphorylase (13). Thus from the standpoint of present knowledge the question of de novo synthesis of glycogen must be left open. Considering the
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P. W. RoBBINS, A. WRIGHT, AND M. DANKERT PolysaccharideBiosynthesis
or other components on or near the cytoplasmic membrane. It has also been
postulated that in the absence of a cell wall the various component macro-
molecules that would make up the cell wall diffuse away from the exterior
surface of the membrane too rapidly to undergo "self-organizing" reactions
such as covalent cross-linking and complex hydrogen bond interactions. This
"diffusion barrier" postulate for the priming function of cell wall in its own
synthesis is attractive, but is not compatible with the reversion of stable L-forms
on hard agar since agar has little effect on the diffusion rate of even rather
large molecules. It is possible that hard agar and hard gelatin change the
conformation of the cytoplasmic membrane and that this conformational
change leads in some as yet undefined way to the organization of the cell
wall macromolecules into a fixed pattern. Certainly it would be useful to
know whether the initiation of cell wall synthesis in the hard agar system
bears any relationship at all to the resumption of cell wall synthesis in re-
verting L-forms, since in one case it is logical to assume that priming is a
physical effect of the medium while in the other case a chemical priming by
wall fragments would seem more reasonable.
The Role of the Cytoplasmic Membrane in PolysaccharideSynthesis
Work with cell-free systems that catalyze the synthesis of heteropolysaccharides
by transfer from nucleoside diphosphate sugar derivatives has suggested that
elements of the cytoplasmic membrane are involved in the synthetic process.
For example, studies by Glaser and Brown (17) with a preparation from
Rous sarcoma cells and the work of Dorfman and coworkers (14) with en-
zymes from Group A Streptococci showed that hyaluronic acid synthesis is
associated with cell particulate fractions. Smith et al. (18) purified the system
that catalyzes the synthesis of Type III Pneumococcus polysaccharide by am-
monium sulfate fractionation and found that although the final preparation
is essentially free of nucleic acid it can be precipitated by ultracentrifugation
and exhibits typical "microsomal" properties. Recent work on the biosyn-
thesis of the lipopolysaccharide component of the cell wall of Gram-negative
bacteria has provided direct evidence for the involvement of membrane
components in complex polysaccharide synthesis. Lipopolysaccharide has a
highly complex structure which, as indicated in Fig. 4, can be subdivided into
three main components: the backbone and the R-specific side chains, which
together constitute the "core," and the smooth O-antigen. The backbone
(19, 20) contains heptose, phosphate, 0-phosphorylethanolamine, and 2-keto-
3-deoxy-octonate (21), which links it to the lipid A of Westphal (20). The
R-specific chains (19) contain glucose, galactose, and N-acetylglucosamine
and are probably joined to the backbone by a glucose-heptose linkage (22).
The O-specific side chains determine the serological groups of enteric bac-
teria and may be linked to the N-acetylglucosamine units of the R-chains
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APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES
(22). Unlike the core material the smooth (^) O-antigen varies widely in mono- saccharide composition. Salmonella groups B and (^) E, which will be discussed below, have repeating sequences containing galactose, mannose, and rham- nose. Mutants that lack the capacity to synthesize UDP-glucose or UDP- galactose (^) form an incomplete lipopolysaccharide that is deficient in both the R-specific and O-specific side chains (23, 24). Such mutants have been used by Rothfield, Osborn, and Horecker (25) to study the incorporation of specific glucosyl or galactosyl residues into the incomplete lipopolysaccharide core.
Heptose Heptose KDO
H-C CH 2 I
o 0
I I 0- 0 CH 2 Glu,Gal, AcGm CH - NH (^3)
' Lipid A
Gal Rh Man
FIGURE 4. Structural components of lipopolysaccharide. The core substance is en- closed by the square brackets and contains 2-keto-3-deoxy-octonate (KDO), heptose, O-phosphorylethanolamine, phosphate, glucose (Glu), galactose (Gal), and N-acetyl- glucosamine (AcGm). The core is linked to lipid A through KDO. The round brackets enclose the repeating sequence of the O-antigen of Salmonella newington which contains galactose, rhamnose (Rh), and mannose (Man). The values of n and m are unknown.
The reactions have been (^) formulated as follows:-
(a) (^) Glucose-deficient lipopolysaccharide + UDP-glucose
- glucosyl-lipopolysaccharide (+ UDP) (b) Glucosyl-lipopolysaccharide + UDP-galactose -- > galactosyl-glucosyl-lipopolysaccharide (+ UDP)
In each case enzyme activity is found in both the cell wall membrane frac- tion and (^) "soluble" fraction derived from sonicated cells. The substrate speci- ficity and the binding of (^) enzyme to its macromolecular substrate have been
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APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES
galactose (Fig. 5), and the former also contains the dideoxyhexose abequose. In each of these cases the presence of membrane material is necessary for synthesis. Weiner et al. (29) and Wright et al. (30) have also demonstrated that the O-antigen chains are not directly derived from the nucleotide sugars but are assembled from trisaccharide repeating units preformed on a lipid intermediate. The sequence of reactions involved in the biosynthesis of the
TDP-Rh [TDP]
UDF
(Man-Rh-Gal)- "Core'^ "Core" FIGURE 6. Scheme for the involvement of lipid intermediates in the^ synthesis^ of^ Sal- monella O-antigen. The system is described in the text. Postulated intermediates and products are shown in square brackets. The following abbreviations are^ used: UDP-Gal, uridine diphosphate-D-galactose TDP-Rh, thymidine diphosphate-L-rhamnose GDP-Man, guanosine diphosphate-D-mannose.
Salmonella O-antigen is^ shown^ in^ Fig.^ 6.^ The^ first^ transfer^ to^ the lipid^ ac- ceptor is that of^ galactose-l-phosphate^ from^ the donor^ UDP-galactose^ with the concurrent release of UMP. UMP inhibits the reaction and can also cause its reversal. The second transfer to the lipid acceptor is that of rhamnose, added to every available galactosyl residue to^ form^ the^ disaccharide^ inter- mediate. In the presence of GDP-mannose the^ trisaccharide^ repeating^ unit^ is formed and immediately converted^ into^ polysaccharide.^ Direct^ evidence^ for the participation of^ the^ trisaccharide^ phosphate^ lipid^ intermediate^ at^ this stage has been provided^ by^ Weiner^ et^ al.^ (29).^ Working^ with^ S.^ typhimurium
340
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P. (^) W. ROBBINS, A. WRIGHT, AND M. DANKERT (^) Polysaccharide Biosynthesis
they isolated and characterized the mannosylrhamnosylgalactose- l-phosphate
lipid. These workers also isolated long O-antigen chains terminated by ga-
lactose-l-phosphate, a finding which is indicative of the formation of a lipid-
linked polysaccharide by a chain-elongating mechanism based on the poly-
CH 2 0H
H NH CO CH 3
L-Ala
D -Glu
L-Lys
D-Ala [
CH20H
I CH$ NH H-C -CH (^3) CO NH 10 HC -C-NH (^2) (CH 2 ) 2 CO NH l 0O HC-(CH 2 ) 3 -CH-NH-C (^) hvVv\V CO HN HC-C O VVVVVVVV CH 3 FIGURE 7. Repeating unit of the mucopeptide (^) of Gram-positive bacteria. The poly- saccharide backbone has alternating (^) units of N-acetylglucosamine and N-acetylmuramic acid. The N-acetylmuramic acid is substituted (^) by a tetrapeptide containing L-alanine, D-isoglutamine, L-lysine, and D-alanine. (^) The zigzag lines indicate the points of attach- ment of interchain bridges.
merization of repeating units provided (^) by the trisaccharide intermediate. The formation of lipopolysaccharide, (^) the final reaction in the sequence, is achieved by a transfer of polysaccharide (^) to the appropriate acceptor in the lipopolysaccharide (^) core. Such a mechanism has important implications (^) since it illustrates how sub- units of the O-antigen, (^) which is exterior to the cell membrane, (^) might be as- sembled from cytoplasmic precursors in the form of lipid-linked (^) intermediates
341
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P. W. ROBBINS, A. WRIGHT, AND M. DANKERT PolysaccharideBiosynthesis
there are in fact a series of related lipids which^ have^ such^ a^ function;^ struc- tural variation of the lipids could provide the specificity necessary for the concurrent formation of the several complex components of the bacterial cell wall. Studies of other membrane-controlled systems, not only in bacteria but in plant and animal cells, should resolve this interesting and important question.
REFERENCES
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APPROACHES TO THE BIOSYNTHESIS OF MACROMOLECULES
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looked and yet they may play a very important role. An example of that has been, I think, provided in the last 2 or 4 years by the work of Roden in Dorfman's labora- tory who has shown that the acid mucopolysaccharides-I don't remember whether hyaluronic acid^ also,^ but^ certainly^ chondroitin^ sulfuric^ acid^ and^ heparin-are^ at- tached to small protein peptides by a handle consisting of one molecule of xylose and two molecules of galactose. So you can have a molecule of about 300,000 which is at- tached to protein by one single molecule of xylose and this xylose is absolutely neces- sary to combine these polysaccharides to the protein. I think that such minute trace constituents in polysaccharides will probably be found to be of much greater im- portance than we at present realize, and that they are prone to be overlooked due to the complex architecture of such polysaccharides as the Salmonella polysaccharides. But I think this problem is one of the most important if, out of the chemistry of the polysaccharides, results of the kind Dr. Luria envisions should eventuate. Dr. Luria: If there are no other questions, we proceed to the last paper in the pro- gram. This really brings us to what I think is the essential problem of building the functional organization of the cell. The problem is how to keep things together in such a way that they are going to work at the right time and at the right place, and only into the kind of relations in which they are going to be useful for the specific purpose of the cell. This leads us to what I would call the biochemistry of the future, that is, the biochemistry of cellular membranes. It is fit that the biochemistry of the future should come last in a symposium of the past. Dr. Kennedy has very kindly agreed to open the future for us in closing the symposium.
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