Characterization of the lipid linkage region and chain length of the cellubiuronic acid capsule of Streptococcus pneumoniae.

The processive reaction mechanisms of beta-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of beta-1,3-Glc and beta-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 mum, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.

A kinetic model for chain length modulation of Streptococcus pneumoniae cellubiuronan capsular polysaccharide by nucleotide sugar donor concentrations.

The chain length of Streptococcus pneumoniae type 3 capsular polysaccharide (cellubiuronic acid) is tightly regulated by the cellubiuronic acid synthase through an assembly process involving a catalytic motif that is potentially conserved over a wide range of related processive beta-glucan synthases. Cellubiuronic acid is initiated on a lipid and is composed of alternating beta-1,3-Glc and beta-1,4-glucuronic acid (GlcUA) linkages. The entire assembly process is carried out by a polypeptide synthase thought to contain a single active site, suggesting that the donor specificity is controlled by the terminal nonreducing sugar in the acceptor subsite. Shortly after initiation, the synthase undergoes an allosteric transition accompanied by the tight binding of the nascent chain via its nonreducing oligosaccharide terminal segment to the carbohydrate acceptor recognition site. The chain length of polysaccharide assembled by recombinant synthase in Escherichia coli membranes was determined by an ejection mechanism that appeared to be a reversal of the allosteric transition of the synthase from the transitory to the fully processive state. The rates of both ejection and transition were shown to be highly sensitive to the concentration of UDP-GlcUA. As the concentration of UDP-GlcUA was increased, both the rate of synthesis and the processive turnover time increased. The product of the processive turnover time and the rate of synthesis predicted a marked increase in polysaccharide chain size (from 50 to 1150 kDa) over a relatively narrow concentration range of 1-11.5 microm UDP-GlcUA. The kinetic model chain length predictions were in close agreement with chemically determined sizes of polysaccharides synthesized at the same UDP-sugar concentrations. The model indicates that translocation occurs following the addition of GlcUA to the chain terminus, whereas UDP-Glc drives chain termination when inadequate levels of UDP-GlcUA are present. In sum, type 3 synthase appears to modulate polysaccharide chain length by functioning as a concentration-dependent kinetic timing device.

Control of capsular polysaccharide chain length by UDP-sugar substrate concentrations in Streptococcus pneumoniae.

Regulation of chain length is essential to the proper functioning of prokaryotic and eukaryotic polysaccharides. Modulation of polymer size by substrate concentration is an attractive but unexplored control mechanism that has been suggested for many polysaccharides. The Streptococcus pneumoniae capsular polysaccharide is essential for virulence, and regulation of its size is critical for survival in different host environments. Synthesis of the type 3 capsule [-4)-beta-d-Glc-(1-3)-beta-d-GlcUA-(1-] from UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) is catalysed by the type 3 synthase, a processive beta-glycosyltransferase, and requires a UDP-Glc dehydrogenase for conversion of UDP-Glc to UDP-GlcUA. Strains containing mutant UDP-Glc dehydrogenases exhibited reduced levels of UDP-GlcUA, along with reductions in total capsule amount and polymer chain length. In both the parent and mutant strains, UDP-Glc levels far exceeded UDP-GlcUA levels, which were very low to undetectable in the absence of blocking synthase activity. The in vivo observations were consistent with in vitro conditions that effect chain termination and ejection of the polysaccharide from the synthase when one substrate is limiting. These data are the first to demonstrate modulation of polysaccharide chain length by substrate concentration and to enable a model for the underlying mechanism. Further, they may have implications for the control of chain length in both prokaryotic and eukaryotic polymers synthesized by similar mechanisms.

Structural studies and mechanism of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase: insights into the initial step of synthesis of dolichyl-phosphate-linked oligosaccharide chains in membranes of endoplasmic reticulum.

Dolichyl-phosphate-mannose (Dol-P-Man) synthase catalyzes the reversible formation of a key intermediate that is involved as a mannosyl donor in at least three different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum (ER), where it transfers mannose from the water soluble cytoplasmic donor, guanosine 5'-diphosphate (GDP)-Man, to the membrane-bound, extremely hydrophobic, and long-chain polyisoprenoid acceptor, dolichyl-phosphate (Dol-P). The enzyme from Saccharomyces cerevisiae has been utilized to investigate the structure and activity of the protein and interactions of the enzyme with Dol-P and synthetic Dol-P analogs containing fluorescent probes. These interactions have been explored utilizing fluorescence resonance energy transfer (FRET) to establish intramolecular distances within the protein molecule as well as intermolecular distances to determine the localization of the active site and the hydrophobic substrate on the enzyme's surface. A three-dimensional (3D) model of the enzyme was produced with bound substrates, Dol-P, GDP-Man, and divalent cations to delineate the binding sites for these substrates as well as the catalytic site. The FRET analysis was used to characterize the functional properties of the enzyme and to evaluate its modeled structure. The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.

Role of the carbohydrate binding site of the Streptococcus pneumoniae capsular polysaccharide type 3 synthase in the transition from oligosaccharide to polysaccharide synthesis.

The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-beta-D-GlcUA-(1, 4)-beta-D-Glc-(1-]n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharylphosphatidylglycerol primer becomes tightly bound to the carbohydrate acceptor recognition site of the synthase. Using the recombinant synthase in Escherichia coli membranes, we determined that a critical oligosaccharide length of approximately 8 monosaccharides was required for recognition of the growing chain by the synthase. Upon binding of the oligosaccharide-lipid to the carbohydrate recognition site, the polymerization reaction entered a highly processive phase to produce polymer of high molecular weight. The initial oligosaccharide-synthetic phase also appeared to be processive, the duration of which was enhanced by the concentration of UDP-GlcUA and diminished by an increase in temperature. The overall reaction approached a steady state equilibrium between the polymer- and oligosaccharide-forming phases that was shifted toward the former by higher UDP-GlcUA levels or lower temperatures and toward the latter by lower concentrations of UDP-GlcUA or higher temperatures. The transition step between the two enzymatic phases demonstrated cooperative kinetics, which is predicted to reflect a possible reorientation of the oligosaccharide-lipid in conjunction with the formation of a tight binding complex.

CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation.

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.

Initiation and synthesis of the Streptococcus pneumoniae type 3 capsule on a phosphatidylglycerol membrane anchor.

The type 3 synthase from Streptococcus pneumoniae is a processive beta-glycosyltransferase that assembles the type 3 polysaccharide [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->] by a multicatalytic process. Polymer synthesis occurs via alternate additions of Glc and GlcUA onto the nonreducing end of the growing polysaccharide chain. In the presence of a single nucleotide sugar substrate, the type 3 synthase ejects its nascent polymer and also adds a single sugar onto a lipid acceptor. Following single sugar incorporation from either UDP-[(14)C]Glc or UDP-[(14)C]GlcUA, we found that phospholipase D digestion of the Glc-labeled lipid yielded a product larger than a monosaccharide, while digestion of the GlcUA-labeled lipid resulted in a product larger than a disaccharide. These data indicated that the lipid acceptor contained a headgroup and that the order of addition to the lipid acceptor was Glc followed by GlcUA. Higher-molecular-weight product synthesized in vitro was also sensitive to phospholipase D digestion, suggesting that the same lipid acceptor was being used for single sugar additions and for polymer formation. Mass spectral analysis of the anionic lipids of a type 3 S. pneumoniae strain demonstrated the presence of glycosylated phosphatidylglycerol. This lipid was also observed in Escherichia coli strains expressing the recombinant type 3 synthase. The presence of the lipid primer in S. pneumoniae membranes explained both the ability of the synthase to reinitiate polysaccharide synthesis following ejection of its nascent chain and the association of newly synthesized polymer with the membrane. Unlike most S. pneumoniae capsular polysaccharides, the type 3 capsule is not covalently linked to the cell wall. The present data indicate that phosphatidylglycerol may anchor the type 3 polysaccharide to the cell membrane.