Glycosyltransferase Complex Research Articles

Neurexin facilitates glycosylation of Dystroglycan to sustain muscle architecture and function in Drosophila

Neurexin, a molecule associated with autism spectrum disorders, is thought to function mainly in neurons. Recently, it was reported that Neurexin is also present in muscle, but the role of Neurexin in muscle is still poorly understood. Here, we demonstrate that the overexpression of Neurexin in muscles effectively restored the locomotor function of Drosophila neurexin mutants, while rescuing effects are observed within the nervous. Notably, the defects in muscle structure and function caused by Neurexin deficiency were similar to those caused by mutations in dystroglycan, a gene associated with progressive muscular dystrophy. The absence of Neurexin leads to muscle attachment defects, emphasizing the essential role of Neurexin in muscle integrity. Furthermore, Neurexin deficiency reduces Dystroglycan glycosylation on the cell surface, which is crucial for maintaining proper muscle structure and function. Finally, Neurexin guides Dystroglycan to the glycosyltransferase complex through interactions with Rotated Abdomen, a homolog of mammalian POMT1. Our findings reveal that Neurexin mediates muscle development and function through Dystroglycan glycosylation, suggesting a potential association between autism spectrum disorders and muscular dystrophy.

Termination of Poly-N-acetylglucosamine (PNAG) Polymerization with N-Acetylglucosamine Analogues.

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In E. coli, the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro, the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

GtfA Interacting with GtfB is Required for PsrP Glycosylation in Streptococcus pneumoniae

Background: Streptococcus pneumoniae can adhere to lung cells via many adhesins, including pneumococcal serine-rich repeat protein (PsrP). Glycosyltransferase A (GtfA) and glycoslytransferase B (GtfB) were found to initiate PsrP glycosylation in Streptococcus pneumoniae serotype 4, isolate TIGR4. Objectives: To test whether GtfA and GtfB are conserved among S. pneumoniae isolates presenting with different serotypes and are involved in the glycosylation of PsrP via formation of the glycosyltransferase complex GtfA-GtfB. Methods: Serotyping of S. pneumoniae was performed using polymerase chain reaction (PCR) assays. The evolutionary divergences of GtfA and GtfB among different isolates were estimated using MEGA4 software. The contribution of GtfA and GtfB to the glycosylation of PsrP was identified using an in vivo glycosylation system in Escherichia coli. Interaction of GtfA with GtfB was assessed using a glutathione S-transferase (GST) pull-down assay and yeast two-hybrid experiment. Results: Nine different S. pneumoniae serotypes were found, of which the most frequent was 19F. The pneumococcal isolate TIGR4 and 22 other clinical isolates were included to estimate evolutionary divergence. The average homologies of the GtfA and GtfB sequences were 99.59% and 98.53%, respectively. Glycosyltransferase A and GtfB were shown to be required for the transfer of N-acetylglucosamine moieties to PsrP1-374. Glutathione S-transferase pull-down assays and yeast two-hybrid experiments showed that GtfA directly interacted with GtfB. Conclusions: Glycosyltransferase A and GtfB are conserved in S. pneumoniae isolates. Glycosyltransferase A interacts with GtfB, forming the glycosyltransferase complex GtfA-GtfB, which is required for PsrP glycosylation.

Glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized.

Glycosylation is the most common and complex cellular modification of proteins and lipids. It is critical for multicellular life and its abrogation often leads to a devastating disease. Yet, the underlying mechanistic details of glycosylation in both health and disease remain unclear. Partly, this is due to the complexity and dynamicity of glycan modifications, and the fact that not all the players are taken into account. Since late 1960s, a vast number of studies have demonstrated that glycosyltransferases typically form homomeric and heteromeric complexes with each other in yeast, plant and animal cells. To propagate their acceptance, we will summarize here accumulated data for their prevalence and potential functional importance for glycosylation focusing mainly on their mutual interactions, the protein domains mediating these interactions, and enzymatic activity changes that occur upon complex formation. Finally, we will highlight the few existing 3D structures of these enzyme complexes to pinpoint their individual nature and to emphasize that their lack is the main obstacle for more detailed understanding of how these enzyme complexes interact and function in a eukaryotic cell.

Structure of pneumococcal GtfA reveals a novel prokaryotic O-GlcNAc transferase

Protein glycosylation is increasingly recognized as an important process for bacterial physiology and pathophysiology. Glycosylation of serine-rich repeat (SRR) glycoprotein PsrP is essential for the pathogenesis of Streptococcus pneumoniae, one of the most common human pathogens. This glycosylation process is initiated by a glycosyltransferase complex comprising two components, a core enzyme GtfA and a co-activator GtfB. Here we report the 2.0-Å crystal structure of GtfA in complex with GlcNAc and UDP. The structure possesses a core domain of GT-B fold and an unprecedented "add-on" domain of DUF1975, which adopts a β-meander structure. This novel DUF1975 domain is critical for the intact glycosyltransferase activity of GtfA–GtfB complex via mediating their self-recognition and the binding to the acceptor protein PsrP. The glycoproteomic analysis revealed a novel pattern of protein O-linked glycosylation at the serine residue cluster. The findings suggest that GtfA is a new glycosyltransferase and provide a structural basis for the future design of inhibitors against the biogenesis of bacterial SRRPs.

Influence of terpenoids and acarbose on glycosyltransferases produced by strain Leuconostoc mesenteroides URE 13

A study of the influence of different plant terpenoids and amino sugar derivate acarbose on the activity of glycosyltransferase complex and purified dextransucrase from Leuconostoc mesenteroides URE 13 strain was carried out. All the tested terpenoids showed an inhibitory effect on glycosyltransferases from strain URE 13 at concentration 0.34 mmol. Out of all studied diterpenoids splendidin showed the strongest inhibitory effect decreasing the activity of both glycosyltransferase complex and dextransucrase with 70% and 90%, respectively. The triterpenoid ursolic showed the second strongest inhibitory effect as the enzyme complex and dextransucrase from strain URE 13 retain 27% and 13% of their initial enzyme activity. Despite the higher degree of inhibition of purified dextransucrase, compared to the enzyme complex, a complete inhibition of the enzyme was not observed at the highest used terpenoid concentration (3.42 mmol). When acarbose was used as an inhibitor, a complete inhibition of dextransucrase was observed at concentration of 6.9 mmol, while the enzyme complex retained 8% of its enzyme activity. Ki values of 0.28 mmol for splendidin, 0.37 mmol for ursolic acid and 0.29 mmol for acarbose were determined from the kinetic studies of purified dextransucrase.

Glycosyltransferase complexes improve glycolipid synthesis

The synthesis of gangliosides GM3 and GD3 is carried out by the successive addition of sialic acid residues on lactosylceramide (LacCer) by the Golgi located sialyltransferases Sial-T1 and Sial-T2, respectively. CHO-K1 cells lack Sial-T2 and only express GM3. Here we show that the activity of Sial-T1 was near 2.5-fold higher in homogenates of CHO-K1 cells transfected to express Sial-T2 (CHO-K1Sial-T2) than in untransfected cells. The appearance of Sial-T1 enzyme or gene transcription activators or the stabilization of the Sial-T1 protein were discarded as possible causes of the activation. Sial-T2 lacking the catalytic domain failed to promote Sial-T1 activation. Since Gal-T1, Sial-T1 and Sial-T2 form a multienzyme complex, we propose that transformation of formed GM3 into GD3 and GT3 by Sial-T2 in the complex leaves Sial-T1 unoccupied, enabled for new rounds of LacCer utilization, which results in its apparent activation.

Spatial Organization and Stoichiometry of N-Terminal Domain-Mediated Glycosyltransferase Complexes in Golgi Membranes Determined by Fret Microscopy

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.

ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta

Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

D-galactan I is a polysaccharide with the disaccharide repeat unit structure [-->3-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]. This glycan represents the lipopolysaccharide O antigen found in many Gram-negative bacteria, including several Klebsiella pneumoniae O serotypes. The polysaccharide is synthesized in the cytoplasm prior to its export via an ATP-binding cassette transporter. Sequence analysis predicts three galactosyltransferases in the D-galactan I genetic locus. They are WbbO (belonging to glycosyltransferase (GT) family 4), WbbM (GT-family 8), and WbbN (GT-family 2). The WbbO and WbbM proteins are each predicted to contain two domains, with the GT modules located toward their C termini. The N-terminal domains of WbbO and WbbM exhibit no similarity to proteins with known function. In vivo complementation assays suggest that all three glycosyltransferases are required for D-galactan I biosynthesis. Using a bacterial two-hybrid system and confirmatory co-purification strategies, evidence is provided for protein-protein interactions among the glycosyltransferases, creating a membrane-located enzyme complex dedicated to d-galactan I biosynthesis.

Modulation of GalT1 and SialT1 Sub-Golgi Localization by SialT2 Expression Reveals an Organellar Level of Glycolipid Synthesis Control

Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.

Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases.

Three key regulatory enzymes in ganglioside biosynthesis, sialyltransferase I (ST1), sialyltransferase II (ST2), and N-acetylgalactosaminyltransferase I (GalNAcT), have been expressed as fusion proteins with green, yellow, or red fluorescent protein (GFP, YFP, or RFP) in F-11A cells. F-11A cells are a substrain of murine neuroblastoma F-11 cells that contain only low endogenous ST2 and GalNAcT activity. The subcellular localization of the fusion proteins has been determined by fluorescence microscopy, and the ganglioside composition of these cells was analyzed by high-performance thin-layer chromatography (HPTLC). ST2-GFP (85 kDa) shows a distinct Golgi localization, whereas ST1-YFP (85 kDa) and GalNAcT-RFP (115 kDa) are broadly distributed in ER and Golgi. Untransfected F-11A cells contain mainly GM3, whereas stable transfection with ST2 or GalNAcT results in the predominant expression of b-series complex gangliosides (BCGs). This result indicates that the expression of ST2 enhances the activity of endogenous GalNAcT and vice versa. The specificity of this reaction has been verified by in vitro activity assays with detergent-solubilized enzymes, suggesting the formation of an enzyme complex between ST2 and GalNAcT but not with ST1. Complex formation has also been verified by co-immunoprecipitation of ST2-GFP upon transient transfection with GalNAcT-HA-RFP and by GFP-to-RFP FRET signals that are confined to the Golgi. FRET analysis also suggests that ST2-GFP binds tightly to pyrene-labeled GM3 but not to ST1. We hypothesize that an ST2-GM3 complex is associated with GalNAcT, resulting in the enhanced conversion of GM3 to GD3 and BCGs in the Golgi. Taken together, our results support the concept that ganglioside biosynthesis is tightly regulated by the formation of glycosyltransferase complexes in the ER and/or Golgi.

Transgenic Expression of the EXT2 Gene in Developing Chondrocytes Enhances the Synthesis of Heparan Sulfate and Bone Formation in Mice

Hereditary multiple exostoses (HME), a dominantly inherited disorder characterized by multiple cartilaginous tumors, is caused by mutations in the gene for, EXT1 or EXT2. Recent studies have revealed that EXT1 and EXT2 are required for the biosynthesis of heparan sulfate and exert maximal transferase activity as a complex. The Drosophila homologue of EXT1 (tout-velu) regulates the movement and signaling of Hedgehog protein, which plays an important role in the regulation of chondrocyte differentiation and bone development. In this study, to investigate the biological role of EXT2 in bone development in vivo and the pathological role of HME mutations in the development of exostoses, we generated transgenic mice expressing EXT2 or mutant EXT2 in developing chondrocytes. Histological analyses and micro-CT scanning showed that the biosynthesis of heparan sulfate and the formation of trabeculae were upregulated in EXT2-transgenic mice, but not in mutant EXT2-transgenic mice. The expression of EXT1 is concomitantly upregulated in EXT2-transgenic and even mutant EXT2-transgenic mice, suggesting an interactive regulation of EXT1 and EXT2 expression. These findings support that the EXT2 gene encodes an essential component of the glycosyltransferase complex required for the biosynthesis of heparan sulfate, which may eventually modulate the signaling involved in bone formation.

Structure of bovine alpha-1,3-galactosyltransferase and its complexes with UDP and DPGal inferred from molecular modeling.

A homology model of alpha-1,3-galactosyltransferase (alpha-1,3-GalT), the retaining enzyme responsible for the formation of alpha-galactosyl epitopes, has been developed by means of molecular modeling using the SpsA glycosyltransferase structure. A protein-ligand docking approach was used to model alpha-1,3-GalT complexed with UDP and UDP-Gal. The comparison of structural features found in the alpha-1,3-GalT homology model with available structural data on this class of enzymes revealed similarities in the UDP-binding pocket. In the predicted structure of the complexes, the pyrophosphate interacts with the DVD motif (Asp-225, Val-226, and Asp-227) of alpha-1,3-GalT through the Mn(2+) cation. The uridine part of the UDP binds into the well-defined cavity that consists of Phe-134, Tyr-139, Ile-140, Val-136, Arg-194, Arg-202, Lys-209, Asp-173, His-218, and Thr-137 in a conformation that is generally observed in the crystal structures of other glycosyltransferase complexes.

EXT genes are differentially expressed in bone and cartilage during mouse embryogenesis

Hereditary multiple exostoses (HME) is a genetically heterogeneous disease characterized by the development of bony protuberances at the ends of all long bones. Genetic analyses have revealed HME to be a multigenic disorder linked to three loci on chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1 and EXT2 genes have been cloned and defined as glycosyltransferases involved in the synthesis of heparan sulfate. EST database analysis has demonstrated additional gene family members, EXT-like genes (EXTL1, EXTL2, and EXTL3), not associated with a HME locus. The mouse homologs of EXT1 and EXT2 have also been cloned and shown to be 99% and 95% identical to their human counterparts, respectively. Here, we report the identification of the mouse EXTL1 gene and show it is 74% identical to the human EXTL1 gene. Expression studies of all three mouse EXT genes throughout various stages of embryonic development were carried out and whole-mount in situ hybridization in the developing limb buds showed high levels of expression of all three EXT genes. However, in situ hybridization of sectioned embryos revealed remarkable differences in expression profiles of EXT1, EXT2, and EXTL1. The identical expression patterns found for the EXT1 and EXT2 genes support the recent observation that both proteins form a glycosyltransferase complex. We suggest a model for exostoses formation based on the involvement of EXT1 and EXT2 in the Indian hedgehog/parathyroid hormone-related peptide (PTHrP) signaling pathway, an important regulator of the chondrocyte maturation process. Dev Dyn 2000;218:452–464. © 2000 Wiley-Liss, Inc.

EXT genes are differentially expressed in bone and cartilage during mouse embryogenesis.

Hereditary multiple exostoses (HME) is a genetically heterogeneous disease characterized by the development of bony protuberances at the ends of all long bones. Genetic analyses have revealed HME to be a multigenic disorder linked to three loci on chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1 and EXT2 genes have been cloned and defined as glycosyltransferases involved in the synthesis of heparan sulfate. EST database analysis has demonstrated additional gene family members, EXT-like genes (EXTL1, EXTL2, and EXTL3), not associated with a HME locus. The mouse homologs of EXT1 and EXT2 have also been cloned and shown to be 99% and 95% identical to their human counterparts, respectively. Here, we report the identification of the mouse EXTL1 gene and show it is 74% identical to the human EXTL1 gene. Expression studies of all three mouse EXT genes throughout various stages of embryonic development were carried out and whole-mount in situ hybridization in the developing limb buds showed high levels of expression of all three EXT genes. However, in situ hybridization of sectioned embryos revealed remarkable differences in expression profiles of EXT1, EXT2, and EXTL1. The identical expression patterns found for the EXT1 and EXT2 genes support the recent observation that both proteins form a glycosyltransferase complex. We suggest a model for exostoses formation based on the involvement of EXT1 and EXT2 in the Indian hedgehog/parathyroid hormone-related peptide (PTHrP) signaling pathway, an important regulator of the chondrocyte maturation process.

Regulation of glycoprotein biosynthesis by formation of specific glycosyltransferase complexes.

Eukaryotic surfaces simultaneously express a complex array of protein-linked oligosaccharides and, during differentiation and on neoplastic transformation, these arrays change. The oligosaccharides linked to asparagine residues of proteins are a complex family of structures that are derived from a common precursor oligosaccharide by a network of competing biosynthetic pathways. The glycosylation reactions that make up these biosynthetic pathways often share common intermediates, requiring the competition between rival glycosyltransferases to be regulated at the supramolecular level. Investigation of two sequential glycosyltransferases that together add N-acetyllactosamine to glycoproteins has revealed their ability to form specific complexes. The functional consequences of complex formation were assessed by investigating the coupled reaction carried out by these sequential enzymes. The membrane enzymes are readily adsorbed by preformed liposomes, and their ability to interact after liposome adsorption has allowed direct investigation of the action carried out by these sequential enzymes when they are coadsorbed to the same liposome or adsorbed to separate liposomes shows the preferential use of endogenously generated intermediate over exogenously added glycoproteins. This facilitated passage of the intermediate glycosylated glycoprotein within the complex results in a tight coupling of the sequential enzymes. The formation of such complexes by sequential glycosyltransferases is proposed as a mechanism for controlling the competition between potentially rival glycosylation sequences during glycoprotein synthesis. The formation of different specific complexes under different conditions provides a flexible mechanism for regulating the synthesis of cell surface glycoproteins during development.

Resolution of the glycosyltransferase activities from two strains of Streptococcus mutans by polyacrylamide gel electrophoresis in the presence of tween 80

The glycosyltransferase complex from Streptococcus mutans can be resolved by polyacrylamide gel electrophoresis following treatment with Tween 80. The enzyme complex was treated with Tween 80 and electrophoresis was performed in the presence of Tween 80. Discrete bands of glycosyltransferase activity were observed when the gels were incubated in buffered sucrose. The enzyme aggregation that occurs with the exocellular glycosyltransferase may involve lipophilic interactions that are disrupted by the Tween 80.

α- D-Glucopyranosyl flouride as a D-glucopyranosyl donor for a glycosyltransferase complex from streptococcus mutans FA1

α- D-Glucopyranosyl fluoride can serve as the D-glucopyranosyl donor for the glycosyltransferase from Streptococcus mutans. The products of the reaction are a D-glucan of high molecular weight and fluoride ion. The rate of reaction was measured by an electrode specific for the fluoride ion. The reaction was inhibited by the substrate at concentrations >30m M, but was not affected by fluoride ion. There was little inhibition of the reaction by a series of monosaccharides, except for 2-amino-2-deoxy D-glucose, D-mannitol, and 3-deoxy- D- ribo-hexose. Maltose, isomaltose, isomaltotriose, and D-fructose all stimulated the release of fluoride from α- D-glucopyranosyl fluoride.

Purification and characterization of a glycosyltransferase complex from the culture broth of Streptococcus mutans FA1

The extracellular glycosyltransferases from Streptococcus mutans FA1 were purified by using the following procedures: ammonium sulfate precipitation, poly(acrylamide) gel filtration, DEAE-cellulose chromatography, and agarose-gel filtration. The dextransucrase and levansucrase activities were purified 350- and 500-fold, respectively, and the ratio of the two activities remained almost constant throughout the purification. Both enzymes have a pH optimum of 6.0, a K m for sucrose of 55mM, and isoelectric points of 3.7 and 4.6. The enzymes are inactivated by repeated freezing and thawing, but retain partial activity even after heating at 100°. The enzyme preparation contains a carbohydrate moiety which does not appear to be either bound levan or dextran.