Mannuronan C-5-epimerases and their application for in vitro and in vivo design of new alginates useful in biotechnology

The industrially important polysaccharide alginate is a linear copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). It is produced commercially by extraction from brown seaweeds, although some of the bacteria belonging to the genera Azotobacter and Pseudomonas also synthesize alginates. Alginates are synthesized as mannuronan, and varying amounts of the M residues in the polymer are then epimerized to G residues by mannuronan C-5-epimerases. The gel-forming, water-binding, and immunogenic properties of the polymer are dependent on the relative amount and sequence distribution of M and G residues. A family of seven calcium-dependent, secreted epimerases (AlgE1-7) from Azotobacter vinelandii have now been characterized, and in this paper the properties of all these enzymes are described. AlgE4 introduces alternating M and G residues into its substrate, while the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences. Two of the enzymes, AlgE1 and AlgE3, are composed of two catalytically active domains, each introducing different G residue sequence patterns in alginate. These results indicate that the enzymes can be used for production of alginates with specialized properties.     

Mode of action and subsite studies of the guluronan block-forming mannuronan C-5 epimerases AlgE1 and AlgE6

AlgE1, AlgE5 and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by the bacterium Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyse the post-polymerization conversion of beta-D-mannuronic acid (M) residues into alpha-L-guluronic acid residues (G). All enzymes show preference for introducing G-residues neighbouring a pre-existing G. They also have the capacity to convert single M residues flanked by G, thus 'condensing' G-blocks to form almost homopolymeric guluronan. Analysis of the length and distribution of G-blocks based on specific enzyme degradation combined with size-exclusion chromatography, electrospray ionization MS, HPAEC-PAD (high-performance anion-exchange chromatography and pulsed amperometric detection), MALDI (matrix-assisted laser-desorption ionization)-MS and NMR revealed large differences in block length and distribution generated by AlgE1 and AlgE6, probably reflecting their different degree of processivity. When acting on polyMG as substrates, AlgE1 initially forms only long homopolymeric G-blocks >50, while AlgE6 gives shorter blocks with a broader block size distribution. Analyses of the AlgE1 and AlgE6 subsite specificities by the same methodology showed that a mannuronan octamer and heptamer respectively were the minimum substrate chain lengths needed to accommodate enzyme activities. The fourth M residue from the non-reducing end is epimerized first by both enzymes. When acting on MG-oligomers, AlgE1 needed a decamer while AlgE6 an octamer to accommodate activity. By performing FIA (flow injection analysis)-MS on the lyase digests of epimerized and standard MG-oligomers, the M residue in position 5 from the non-reducing end was preferentially attacked by both enzymes, creating an MGMGGG-sequence (underlined and boldface indicate the epimerized residue).     

Properties and action pattern of the recombinant mannuronan C-5-epimerase AlgE2

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca²⁺-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of β- -mannuronic acid residues (M) to - -guluronic acid residues (G) in alginate. AlgE2 has been produced by fermentation with a recombinant strain of Escherichia coli, isolated and partially purified. Epimerization with AlgE2 increased the content of G-residues in different alginates from starting values of 0–45% up to approximately 70%. The new G-residues were mainly present in short blocks. Although G-residues may be introduced next to pre-existing G-residues, AlgE2 was not able to epimerize strictly alternating MG-structures. The epimerization with AlgE2 was greatly affected by the concentration of Ca²⁺. The type of alginate used as substrate affected the reaction rate and the reaction pattern especially at low Ca²⁺ concentration. AlgE2 appears to act by a preferred attack mechanism where the enzyme associates with different sequences in the alginate depending on the concentration of Ca²⁺. During epimerization, AlgE2 occasionally causes cleavage of the alginate chain. The observed frequency corresponds to 1–3 breaks per 1,000 M-units epimerized.     

Mode of action of recombinant Azotobacter vinelandii mannuronan C-5 epimerases AlgE2 and AlgE4.

The enzymes mannuronan C-5 epimerases catalyze conversion of beta-D-mannuronic acid to alpha-L-guluronic acid in alginates at the polymer level and thereby introduce sequences that have functional properties relevant to gelation. The enzymatic conversion by recombinant mannuronan C-5 epimerases AlgE4 and AlgE2 on alginate type substrates with different degree of polymerization and initial low fraction of alpha-L-guluronic acid was investigated. Essentially no enzymatic activity was found for fractionated mannuronan oligomer substrates with an average degree of polymerization, DP(n), less than or equal 6, whereas increasing the DP(n) yielded increased epimerization activity. This indicates that these enzymes have an active site consisting of binding domains for consecutive residues that requires interaction with 7 or more consecutive residues to show enzymatic activity. The experimentally determined kinetics of the reaction, and the residue sequence arrangement introduced by the epimerization, were modeled using Monte Carlo simulation accounting for the various competing intrachain substrates and assuming either a processive mode of action or preferred attack. The comparison between experimental data and simulation results suggests that epimerization by AlgE4 is best described by a processive mode of action, whereas the mode of action of AlgE2 appears to be more difficult to determine.     

The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.     

Identification and characterization of an Azotobacter vinelandii type I secretion system responsible for export of the AlgE-type mannuronan C-5-epimerases

Alginate is a linear copolymer of beta-d-mannuronic acid and its C-5-epimer, alpha-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (< or =2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.     

Mapping enzymatic functionalities of mannuronan C-5 epimerases and their modular units by dynamic force spectroscopy

Alginates are (1→4)-linked structural copolyuronans consisting of β-d-mannuronic acid (M) and its C-5 epimer -l-guluronic acid (G). The residue sequence variation is introduced in a unique postpolymerisation step catalysed by a family of C-5 epimerases named AlgE enzymes. The seven known AlgE’s are composed of two modules, designated A and R, present in different number. The molecular details of the structure–function relationship of these seven epimerases, introducing specific residue sequences, are not understood. In this study, single-molecular pair interactions between alginate and AlgE enzymes were investigated using dynamic force spectroscopy. The AlgE enzymes AlgE4 and AlgE6, the recombinant construct PKA1 composed of A- and R-modules from various AlgE’s, as well as separate R- and A-modules were studied. The strength of the protein–mannuronan interaction, when applying a loading rate of 0.6 nN/s, varied from 73 pN (AlgE4) to 144 pN (A-module). The determined potential width, that is, the distance from the activation barrier to the bound substrate molecule, was 0.23 nm for AlgE4, 0.19 nm for AlgE6 and 0.1 nm for the A-module. No attraction was observed between the R-module and the substrate. The observations indicate that the A-module contains the substrate binding site and that the R-module modulates the enzyme–substrate binding strength. The observed AlgE4-polymer residence times, two orders of magnitude longer than expected from k_cat reported for AlgE4, not observed for PKA1, led us to propose a processive mode of action of AlgE4.     

Single-molecular pair unbinding studies of Mannuronan C-5 epimerase AlgE4 and its polymer substrate

Alginate biosynthesis involves C-5-mannuronan epimerases catalyzing the conversion of beta-D-mannuronic acid to alpha-L-guluronic acid at the polymer level. Mannuronan epimerases are modular enzymes where the various modules yield specific sequential patterns of the converted residues in their polymer products. Here, the interaction between the AlgE4 epimerase and mannuronan is determined by dynamic force spectroscopy. The specific unbinding between molecular pairs of mannuronan and AlgE4 as well as its two modules, A and R, respectively, was studied as a function of force loading rate. The mean protein-mannuronan unbinding forces were determined to be in the range 73-144 pN, depending on the protein, at a loading rate of 0.6 nN/s, and increased with increasing loading rate. The position of the activation barrier was determined to be 0.23 +/- 0.04 nm for the AlgE4 and 0.10 +/- 0.02 nm for its A-module. The lack of interaction observed between the R-module and mannuronan suggest that the A-module contains the binding site for the polymer substrate. The ratio between the epimerase-mannuronan dissociation rate and the catalytic rate for epimerization of single hexose residues suggests a processive mode of action of the AlgE4 epimerase yielding the observed sequence pattern in the uronan associated with the A-module of this enzyme.     

Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.     

Expression of SA5K, a secretion antigen of Mycobacterium tuberculosis, inside human macrophages and in sputum from tuberculosis patients

The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1-7 can be used to improve the properties of the commercially important polysaccharide alginate that is widely used in a variety of products, such as food and pharmaceuticals. Since lactic acid bacteria are generally regarded as safe, they are attractive candidates for production of the epimerases. A. vinelandii genes are GC-rich, in contrast to those of lactic acid bacteria, but we show here that significant expression levels of the epimerase AlgE6 can be obtained in Lactococcus lactis using the nisin-controlled expression system. A 1200-fold induction ratio was obtained resulting in an epimerase activity of 23900 dpm mg(-1) h(-1), using a tritiated alginate substrate. The epimerase was detected by Western blotting and nuclear magnetic resonance spectroscopy analysis of its reaction product showed that the enzyme displayed catalytic properties similar to those produced in Escherichia coli.     

Influence of environmental conditions on the activity of the recombinant mannuronan C-5-epimerase AlgE2

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca(2+)-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) in alginate. AlgE2 had a pH optimum between 6.5 and 7 and a temperature optimum around 55 degrees C. Addition of low molecular weight organic compounds, including buffers, amino acids and osmoprotective compounds, affected the activity of the enzyme. The charge, size and stereochemistry of the added compounds were important. The activity of AlgE2, dissolved in various buffers (same pH), decreased with increasing fraction of positively charged buffer ions. Mono- and divalent metal ions also influenced the activity. When Ca(2+) was omitted only Sr(2+), of the metal ions tested, supported some activity of AlgE2. At high concentration of Ca(2+) (3.3 mM) these ions had a negative effect on the activity, whereas at low Ca(2+) concentration (0.58 mM) the activity was enhanced by addition of Sr(2+), and to some degree also by addition of Mg(2+) and Mn(2+). During epimerization AlgE2 occasionally causes cleavage of the alginate chain. These chain breaks could not be prevented by changes in the conditions during the epimerization. The composition and sequential structure of epimerized alginate was not altered by changes in the epimerization conditions.     

The Pseudomonas syringae genome encodes a combined mannuronan C-5-epimerase and O-acetylhydrolase, which strongly enhances the predicted gel-forming properties of alginates

Alginates are industrially important, linear copolymers of beta-d-mannuronic acid (M) and its C-5-epimer alpha-l-guluronic acid (G). The G residues originate from a postpolymerization reaction catalyzed by mannuronan C-5-epimerases (MEs), leading to extensive variability in M/G ratios and distribution patterns. Alginates containing long continuous stretches of G residues (G blocks) can form strong gels, a polymer type not found in alginate-producing bacteria belonging to the genus Pseudomonas. Here we show that the Pseudomonas syringae genome encodes a Ca(2+)-dependent ME (PsmE) that efficiently forms such G blocks in vitro. The deduced PsmE protein consists of 1610 amino acids and is a modular enzyme related to the previously characterized family of Azotobacter vinelandii ME (AlgE1-7). A- and R-like modules with sequence similarity to those in the AlgE enzymes are found in PsmE, and the A module of PsmE (PsmEA) was found to be sufficient for epimerization. Interestingly, an R module from AlgE4 stimulated Ps-mEA activity. PsmE contains two regions designated M and RTX, both presumably involved in the binding of Ca(2+). Bacterial alginates are partly acetylated, and such modified residues cannot be epimerized. Based on a detailed computer-assisted analysis and experimental studies another PsmE region, designated N, was found to encode an acetylhydrolase. By the combined action of N and A PsmE was capable of redesigning an extensively acetylated alginate low in G from a non gel-forming to a gel-forming state. Such a property has to our knowledge not been previously reported for an enzyme acting on a polysaccharide.     

NMR structure of the R-module: a parallel beta-roll subunit from an Azotobacter vinelandii mannuronan C-5 epimerase

In the bacterium Azotobacter vinelandii, a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7) has been identified. These epimerases are responsible for the epimerization of beta-d-mannuronic acid to alpha-l-guluronic acid in alginate polymers. The epimerases consist of two types of structural modules, designated A (one or two copies) and R (one to seven copies). The structure of the catalytically active A-module from the smallest epimerase AlgE4 (consisting of AR) has been solved recently. This paper describes the NMR structure of the R-module from AlgE4 and its titration with a substrate analogue and paramagnetic thulium ions. The R-module folds into a right-handed parallel beta-roll. The overall shape of the R-module is an elongated molecule with a positively charged patch that interacts with the substrate. Titration of the R-module with thulium indicated possible calcium binding sites in the loops formed by the nonarepeat sequences in the N-terminal part of the molecule and the importance of calcium binding for the stability of the R-module. Structure calculations showed that calcium ions can be incorporated in these loops without structural violations and changes. Based on the structure and the electrostatic surface potential of both the A- and R-module from AlgE4, a model for the appearance of the whole protein is proposed.     

Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii

Alginate is a family of linear copolymers of (1-->4)-linked beta-d-mannuronic acid and its C-5 epimer alpha-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a right-handed parallel beta-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The beta-helix is composed of four parallel beta-sheets, comprising 12 complete turns, and has an amphipathic alpha-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction.     

Characterization of Three New Azotobacter vinelandii Alginate Lyases,One of Which Is Involved in Cyst Germination

Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic acid and α-l-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca²⁺. All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.Azotobacter vinelandii is a nitrogen-fixing bacterium found in soil. A. vinelandii and several species belonging to the related genus Pseudomonas have been found to produce the polymer alginate. This linear, extracellular polysaccharide is composed of 1-4-linked β-d-mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G) (35), and the relative amount and distribution of these two residues vary according to the species and growth conditions. Some of the M residues in bacterial alginates may be O acetylated at C-2, C-3, or both C-2 and C-3 (34).Alginate is first synthesized as mannuronan, and the G residues are introduced by mannuronan C-5 epimerases. All genome-sequenced alginate-producing bacteria have been found to encode a periplasmic epimerase, AlgG, that epimerizes some of the M residues in the polymer into G residues (40). AlgG seems to be unable to epimerize an M residue next to a preexisting G residue in vivo. A. vinelandii also encodes a family of secreted mannuronan C-5 epimerases (AlgE1-7) (40), some of which are able to form stretches of consecutive G residues (G blocks). Alginates containing G blocks can be cross-linked by divalent cations and thereby form gels (35).Polysaccharide lyases (EC 4.2.2.-) are a group of enzymes which cleave the polymer chains via a β-elimination mechanism, resulting in the formation of a double bond at the newly formed nonreducing end. For alginate lyases, 4-deoxy-l-erythro-hex-4-enepyranosyluronate (denoted as Δ) is formed at the nonreducing end. Several such lyases have been purified from both alginate-producing and alginate-degrading organisms, as reviewed by Wong et al. (42). When they are classified according to primary structure, the alginate lyases belong to the polysaccharide-degrading enzyme families PL5, PL6, PL7, PL14, PL17, and PL18 (http://www.cazy.org). Alginate molecules may contain four different bonds (M-M, M-G, G-M, and G-G), and alginate lyases may therefore be classified according to their preferred substrate specificities. It is now possible to obtain pure mannuronan and nearly pure (MG)_n and G blocks (17, 19, 20), and this allows for an improved assessment of the substrate specificities of the alginate lyases.The following two alginate lyases have been characterized in A. vinelandii: the periplasmic AlgL that belongs to the PL5 family (15) and the extracellular bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7 (36, 37). AlgL is encoded by the alginate biosynthesis operon, similar to what has been found in all characterized alginate-producing bacteria. This enzyme cleaves M-M and M-G bonds (15), while AlgE7 preferably degrades G-MM and G-GM bonds (37). The latter enzyme is also able to introduce G residues in the alginate, thus creating the preferred substrate for the lyase.When A. vinelandii experiences a lack of nutrients, it will develop into a dormant cell designated cyst (30). The cell is then protected against desiccation by a multilayered coat, of which gel-forming alginate is a necessary part. Resuspension of cysts in a medium containing glucose leads to a germination process in which vegetative cells eventually escape from the cyst coat. It has been proposed that an alginate lyase may be involved in the rupture of the coat (43). AlgL is dispensable for germination (38), while the biological function of AlgE7 is unknown. In this report, we use the available draft genome sequence of A. vinelandii to identify three additional putative lyases and evaluate their and AlgE7''s role in growth, encystment, and germination of the bacterium.     

Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MG-oligosaccharides by NMR spectroscopy and ESI-mass spectrometry

Alginate with long strictly alternating sequences of mannuronic (M) and guluronic (G) acid residues, F(G) = 0.47 and F(GG) = 0.0, was prepared by incubating mannuronan with the recombinant C-5 epimerase AlgE4. By partial acid hydrolysis of this PolyMG alginate at pH values from 2.8 to 4.5 at 95 degrees C, alpha-L-GulpA-(1-->4)-beta-D-ManpA (G-M) linkages were hydrolyzed far faster than beta-D-ManpA-(1-->4)-alpha-L-GulpA (M-G) linkages in the polymer chain. The ratio of the rates (kG-M/kM-G) decreased with increasing pH. The dominant mechanism for hydrolysis of (1-->4)-linked PolyMG in weak acid was thus proved to be an intramolecular catalysis of glycosidic cleavage of the linkages at C-4 by the undissociated carboxyl groups at C-5 in the respective units. The higher degradation rate of G-M than M-G glycosidic linkages in the polymer chain of MG-alginate at pH 3.5 and 95 degrees C was exploited to make oligomers mainly consisting of M on the nonreducing and G on the reducing end and, thus, a majority of oligomers with an even number of residues. The ratio of the rate constants kG-M/kM-G at this pH was 10.7. The MG-hydrolysate was separated by size exclusion chromatography and the MG oligosaccharide fractions analyzed by electrospray ionization-mass spectrometry together with 1H and 13C NMR spectroscopy. Chemical shifts of MG-oligomers (DP2-DP5) were elucidated by 2D 1H and 13C NMR.     

The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme

The Azotobacter vinelandii genome encodes a family of seven secreted Ca(2+)-dependent epimerases (AlgE1--7) catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. AlgE1--7 are composed of two types of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an alginase, and here we show that the lyase activity is Ca(2+)-dependent and also responds similarly to the epimerases in the presence of other divalent cations. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich alginate from the brown algae Macrocystis pyrifera most effectively, producing oligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly G/MM and/or G/GM. Since G-moieties dominated at the reducing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay between the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5'-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimerase activities. Epimerization and lyase activity are believed to be mechanistically related, and the results reported here strongly support this hypothesis by suggesting that the same enzymatic site can catalyze both reactions.     

C(6)-oxidation followed by C(5)-epimerization of guar gum studied by high field NMR

Guar gum, a beta-D-(1-->4)-linked D-mannan with alpha-D-galactopyranosyl units attached as side groups, was treated with alpha-galactosidase, an enzyme that splits off the alpha-D-galactosyl units to obtain a galactomannan with a low galactose content. The galactose-depleted polysaccharide was then selectively oxidized in C(6) position and epimerized using mannuronan C(5)-epimerases, namely AlgE1, AlgE4, AlgE6, and their mixtures, obtaining new pseudo-alginates. In this paper, we report a full high field 1D and 2D NMR study of guar gum as such and of the galactose-depleted, oxidized and epimerized compounds, respectively. From the 1H NMR spectra, the degree of epimerization, the distribution of mannuronic acid (M) and guluronic acid (G) residues and the average G-block length, N(G>1), were obtained. By means of NMR diffusion experiments, it was also shown that no significant degradation of the polysaccharide occurs as a consequence of the epimerization reactions.     

A high field NMR study of the products ensuing from konjak glucomannan C(6)-oxidation followed by enzymatic C(5)-epimerization

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.