Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAβ1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.     

In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at −80 °C using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/μg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed.     

Structure/function analysis of Pasteurella multocida heparosan synthases: toward defining enzyme specificity and engineering novel catalysts

The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (～65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.     

Chemoenzymatic synthesis with distinct Pasteurella heparosan synthases: monodisperse polymers and unnatural structures

Heparosan (-GlcUA-beta1,4-GlcNAc-alpha1,4-)(n) is a member of the glycosaminoglycan polysaccharide family found in the capsule of certain pathogenic bacteria as well as the precursor for the vertebrate polymers, heparin and heparan sulfate. The two heparosan synthases from the Gram-negative bacteria Pasteurella multocida, PmHS1 and PmHS2, were efficiently expressed and purified using maltose-binding protein fusion constructs. These relatively homologous synthases displayed distinct catalytic characteristics. PmHS1, but not PmHS2, was able to produce large molecular mass (100-800 kDa) monodisperse polymers in synchronized, stoichiometrically controlled reactions in vitro. PmHS2, but not PmHS1, was able to utilize many unnatural UDP-sugar analogs (including substrates with acetamido-containing uronic acids or longer acyl chain hexosamine derivatives) in vitro. Overall these findings reveal potential differences in the active sites of these two Pasteurella enzymes. In the future, these catalysts should allow the creation of a variety of heparosan and heparinoids with utility for medical applications.     

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida,PmHS1

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

     

Identification of a distinct, cryptic heparosan synthase from Pasteurella multocida types A, D, and F

The extracellular polysaccharide capsules of Pasteurella multocida types A, D, and F are composed of hyaluronan, N-acetylheparosan (heparosan or unsulfated, unepimerized heparin), and unsulfated chondroitin, respectively. Previously, a type D heparosan synthase, a glycosyltransferase that forms the repeating disaccharide heparosan backbone, was identified. Here, a approximately 73% identical gene product that is encoded outside of the capsule biosynthesis locus was also shown to be a functional heparosan synthase. Unlike PmHS1, the PmHS2 enzyme was not stimulated greatly by the addition of an exogenous polymer acceptor and yielded smaller- molecular-weight-product size distributions. Virtually identical hssB genes are found in most type A, D, and F isolates. The occurrence of multiple polysaccharide synthases in a single strain invokes the potential for capsular variation.     

Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (beta 4-glucuronic acid-beta 3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (approximately 10-20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (approximately 1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.     

Functional characterization of PmHS1, a Pasteurella multocida heparosan synthase

Heparosan synthase 1 (PmHS1) from Pasteurella multocida Type D is a dual action glycosyltransferase enzyme that transfers monosaccharide units from uridine diphospho (UDP) sugar precursors to form the polysaccharide heparosan (N-acetylheparosan), which is composed of alternating (-alpha4-GlcNAc-beta1,4-GlcUA-1-) repeats. We have used molecular genetic means to remove regions nonessential for catalytic activity from the amino- and the carboxyl-terminal regions as well as characterized the functional regions involved in GlcUA-transferase activity and in GlcNAc-transferase activity. Mutation of either one of the two regions containing aspartate-X-aspartate (DXD) residue-containing motifs resulted in complete or substantial loss of heparosan polymerizing activity. However, certain mutant proteins retained only GlcUA-transferase activity while some constructs possessed only GlcNAc-transferase activity. Therefore, it appears that the PmHS1 polypeptide is composed of two types of glycosyltransferases in a single polypeptide as was found for the Pasteurella multocida Type A PmHAS, the hyaluronan synthase that makes the alternating (-beta3-GlcNAc-beta1,4-GlcUA-1-) polymer. However, there is low amino acid similarity between the PmHAS and PmHS1 enzymes, and the relative placement of the GlcUA-transferase and GlcNAc-transferase domains within the two polypeptides is reversed. Even though the monosaccharide compositions of hyaluronan and heparosan are identical, such differences in the sequences of the catalysts are expected because the PmHAS employs only inverting sugar transfer mechanisms whereas PmHS1 requires both retaining and inverting mechanisms.     

Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity.

The D-glucuronosyl (GlcA)- and N-acetyl-D-galactosaminyl (GalNAc)-transferases involved in chondroitin sulphate biosynthesis were studied in a microsomal preparation from chick-embryo chondrocytes. Transfer of GlcA and GalNAc from their UDP derivatives to 3H-labelled oligosaccharides prepared from chondroitin sulphate and hyaluronic acid was assayed by h.p.l.c. of the reaction mixture. Conditions required for maximal activities of the two enzymes were remarkably similar. Activities were stimulated 3.5-6-fold by neutral detergents. Both enzymes were completely inhibited by EDTA and maximally stimulated by MnCl2 or CoCl2. MgCl2 neither stimulated nor inhibited. The GlcA transferase showed a sharp pH optimum between pH5 and 6, whereas the GalNAc transferase gave a broad optimum from pH 5 to 8. At pH 7 under optimal conditions, the GalNAc transferase gave a velocity that was twice that of the GlcA transferase. Oligosaccharides prepared from chondroitin 4-sulphate and hyaluronic acid were almost inactive as acceptors for both enzymes, whereas oligosaccharides from chondroitin 6-sulphate and chondroitin gave similar rates that were 70-80-fold higher than those observed with the endogenous acceptors. Oligosaccharide acceptors with degrees of polymerization of 6 or higher gave similar Km and Vmax. values, but the smaller oligosaccharides were less effective acceptors. These results are discussed in terms of the implications for regulation of the overall rates of the chain-elongation fractions in chondroitin sulphate synthesis in vivo.     

Structure and Taste of Some Analogs of Naringin

The structures of acidic oligosaccharides synthesized by a transglycosylation reaction by Bacillus circulans β-galactosidase, using lactose as the galactosyl donor, and N-acetylneuraminic acid (NeuAc) and glucuronic acid (GlcUA) as the acceptors were investigated. Acidic oligosaccharides thus synthesized were purified by anion exchange chromatography and charcoal chromatography. The MS and NMR studies indicated that the acidic oligosaccharides from NeuAc were Galβ-(1→8)-NeuAc, Galβ-(1→9)-NeuAc, and Galβ-(1→3)-Galβ-(1→8)-NeuAc, and those from GlcUA were Galβ-(1→3)-GlcUA and Galβ-(1→4)-Galβ-(1→3)-GlcUA. These are novel acidic galactooligosaccharides.     

Heparosan Chain Characterization: Sequential Depolymerization of E. Coli K5 Heparosan by a Bacterial Eliminase Heparin Lyase III and a Bacterial Hydrolase Heparanase Bp to Prepare Defined Oligomers

Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep-3, an eliminase from Flavobacterium heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d -glucuronic acid (GlcA) and N-acetyl-d -glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.     

Regulation of I-branched poly-N-acetyllactosamine synthesis. Concerted actions by I-extension enzyme, I-branching enzyme, and beta1,4-galactosyltransferase I

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching beta1, 6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewisx, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta1, 6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers beta1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various beta1, 4-galactosyltransferases (beta4Gal-Ts), beta4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a beta1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of beta4Gal-TI and i-extension beta1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Galbeta1-->4GlcNAcbeta1-->3 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that beta4Gal-TI adds a galactose poorly to beta1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while beta1, 3-N-acetylglucosaminyltransferase and beta4Gal-TI efficiently add N-acetyllactosamine to linear poly-N-acetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.     

Controlled Photochemical Depolymerization of K5 Heparosan, a Bioengineered Heparin Precursor

Heparosan is a polysaccharide, which serves as the critical precursor in heparin biosynthesis and chemoenzymatic synthesis of bioengineered heparin. Because the molecular weight of microbial heparosan is considerably larger than heparin, the controlled depolymerization of microbial heparosan is necessary prior to its conversion to bioengineered heparin. We have previously reported that other acidic polysaccharides could be partially depolymerized with maintenance of their internal structure using a titanium dioxide-catalyzed photochemical reaction. This photolytic process is characterized by the generation of reactive oxygen species that oxidize individual saccharide residues within the polysaccharide chain. Using a similar approach, a microbial heparosan from Escherichia coli K5 of molecular weight >15,000 was depolymerized to a heparosan of molecular weight 8,000. The (1)H-NMR spectra obtained showed that the photolyzed heparosan maintained the same structure as the starting heparosan. The polysaccharide chains of the photochemically depolymerized heparosan were also characterized by electrospray ionization-Fourier-transform mass spectrometry. While the chain of K5 heparosan starting material contained primarily an even number of saccharide residues, as a result of coliphage K5 lyase processing, both odd and even chain numbers were detected in the photochemically-depolymerized heparosan. These results suggest that the photochemical depolymerization of heparosan was a random process that can take place at either the glucuronic acid or the N-acetylglucosamine residue within the heparosan polysaccharide.     

In vitro heparan sulfate polymerization: crucial roles of core protein moieties of primer substrates in addition to the EXT1-EXT2 interaction

Heparan, the common unsulfated precursor of heparan sulfate (HS) and heparin, is synthesized on the glycosaminoglycan-protein linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl attached to the respective core proteins presumably by HS co-polymerases encoded by EXT1 and EXT2, the genetic defects of which result in hereditary multiple exostoses in humans. Although both EXT1 and EXT2 exhibit GlcNAc transferase and GlcUA transferase activities required for the HS synthesis, no HS chain polymerization has been demonstrated in vitro using recombinant enzymes. Here we report in vitro HS polymerization. Recombinant soluble enzymes expressed by co-transfection of EXT1 and EXT2 synthesized heparan polymers with average molecular weights greater than 1.7 x 105 using UDP-[3H]GlcNAc and UDP-GlcUA as donors on the recombinant glypican-1 core protein and also on the synthetic linkage region analog GlcUA-Gal-O-C2H4NH-benzyloxycarbonyl. Moreover, in our in vitro polymerization system, a part time proteoglycan, alpha-thrombomodulin, that is normally modified with chondroitin sulfate served as a polymerization primer for heparan chain. In contrast, no polymerization was achieved with a mixture of individually expressed EXT1 and EXT2 or with acceptor substrates such as N-acetylheparosan oligosaccharides or the linkage region tetrasaccharide-Ser, which are devoid of a hydrophobic aglycon, suggesting the critical requirement of core protein moieties in addition to the interaction between EXT1 and EXT2 for HS polymerization.     

Glycolipid glycosyl transferases of a hamster cell line in culture. II. Subcellular distribution and the effect of culture age and density.

The activities of two galactosyl transferases catalysing the formation of di- and tri-glycosyl ceramides in NIL-2 hamster cells have been studied with respect to culture age and density, subcellular distribution, and transformation of cells by virus. The activity of the transferases was found to increase considerably as culture density increased, although maximal activities were found before appreciable cell contact occurred. The highest transferase activities were found in the endoplasmic reticulum. Virus transformation reduces the activity of the transferase catalysing triglycosyl ceramide synthesis, while the transferase catalysing diglycosyl ceramide synthesis is slightly increased. There is no evidence that the transformed cells produce a dialysable soluble inhibitor of transferase activities.     

Structural analysis of α1,3-linked galactose-containing oligosaccharides in Schizosaccharomyces pombe mutants harboring single and multiple α-galactosyltransferase genes disruptions

In the fission yeast Schizosaccharomyces pombe, galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases in the lumen of the Golgi apparatus. In S. pombe, the major in vitro α1,2-galactosyltransferase activity has been purified, the gma12(+) gene has been cloned, and three α-galactosyltransferase genes (gmh1(+)-gmh3(+)) have also been partially characterized. In this study, we found three additional uncharacterized genes with homology to gmh1(+) (gmh4(+)-gmh6(+)) in the fission yeast genome sequence. All possible single disruption mutants and the septuple disruption strain were constructed and characterized. The electrophoretic mobility of acid phosphatase prepared from gma12Δ, gmh2Δ, gmh3Δ and gmh6Δ mutants was higher than that from wild type, indicating that Gma12p, Gmh2p, Gmh3p and Gmh6p are required for the galactosylation of N-linked oligosaccharides. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides from each single mutant showed that Gma12p, Gmh2p and Gmh6p are involved in galactosylation of O-linked oligosaccharides. The septuple mutant exhibited similar drug and temperature sensitivity as a gms1Δ mutant that is incapable of galactosylation. Oligosaccharide structural analysis based on HPLC and methylation analysis revealed that the septuple mutant still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating that an unknown α1,3-galactosyltransferase activity was still present in the septuple mutant.     

Single-step purification and h.p.l.c. analysis of glutathione transferase 8-8 in rat tissues.

GSSG selectively elutes two GSH transferases from a mixture of rat GSH transferases bound to a GSH-agarose affinity matrix. One is a form of GSH transferase 1-1 and the other is shown to be GSH transferase 8-8. By using tissues that lack this form of GSH transferase 1-1 (e.g. lung), GSH transferase 8-8 may thus be purified from cytosol in a single step. Quantitative analysis of the tissue distribution of GSH transferase 8-8 was obtained by h.p.l.c.     

Chaperone-assisted expression of KfiC glucuronyltransferase from <Emphasis Type="Italic">Escherichia coli</Emphasis> K5 leads to heparosan production in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 in absence of the stabilisator KfiB

The heparosan synthase of Escherichia coli K5 is composed of the glycosyltransferases KfiA and KfiC which synthesize the polysaccharide heparosan (N-acetylheparosan). A third protein, KfiB, is required to stabilize the KfiAC complex in the bacteria and to transport this complex to the inner membrane where the initiation of polymerization occurs. In this report, we fused KfiC with the E. coli trigger factor (TF) to stabilize KfiC, thus activating the enzyme in the absence of KfiB. Different recombinant plasmids were constructed to compare the impact of the presence or absence of KfiB and the presence of the trigger factor as a fusion protein. Several E. coli BL21-derived strains were transformed with recombinant plasmids and cultivated in fed-batch conditions on minimal medium. The bTCA strain overexpressing fused TF-KfiC together with KfiA and KfiD, but lacking KfiB produced 1.5 g/L of total heparosan after 24 h of fed-batch cultivation. This heparosan was essentially intracellular early in the culture, providing evidence that KfiB primarily plays a role in the exportation process. However, over time, heparosan became mostly extracellular, likely due to passive diffusion or partial cell disruption upon product accumulation.     

Identity of malonyl and palmitoyl transferase of fatty acid synthetase from yeast. Functional interrelationships between the acyl transferases.

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4--5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.     

Biosynthesis of yeast mannoproteins. Synthesis of mannan outer chain and of dolichol derivatives.

The Saccharomyces cerevisiae mutants affected in the structure of mannan outer chain were found to synthesize dolichol diphosphate-linked oligosaccharides identical in size to those of the wild type strain. The mannosyl transferases involved in the synthesis of the outer chain had an absolute requirement for manganese ions and were activated when enzymatic preparations were stored at 2 degrees C, whereas the transferases responsible for the formation of dolichol monophosphate mannose and dolichol diphosphate oligosaccharides were drastically inactivated from the onset of storage and required magnesium or manganese ions, the former being more effective than the latter. Both sets of enzymes could be separated by ion exchange chromatography. In vitro conditions that enhanced the synthesis of dolichol monophosphate mannose did not stimulate the incorporation of mannose residues into the outer chain. It is concluded that dolichol monophosphate mannose is not an intermediate in the synthesis of the outer chain and that this part of mannan and the dolichol diphosphate oligosaccharides are synthesized by different mannosyltransferases.