Mannuronan C-5 epimerase activity in protoplasts of Laminaria digitata

Biosynthesis of alginate in algae may be studied by following the cell wall regeneration of brown seaweed protoplasts in culture. The enzyme mannuronan C-5 epimerase will control the composition of the alginate being synthetized.Freshly isolated protoplasts from the thallus of young Laminaria digitata plants showed only low expression of this enzyme. However, after prolonged periods in culture, this activity increased 15-fold. The synthesis of C-5 epimerase by the protoplasts is probably essential for the formation of a new cell wall.After cellular disruption by osmotic shock and centrifugation, most of the epimerase activity resided in the pellet fraction. This may indicate that the enzyme is membrane associated.     

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).     

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.     

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.     

Mannuronan C-5 epimerases and cellular differentiation of Azotobacter vinelandii

Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a co-polymer of β- d -mannuronic acid (M) and α- l -guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the epimerase-encoded R-modules. This gene is located within the epimerase gene cluster of Azotobacter vinelandii .     

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.     

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.     

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.     

Mucoid strains of Pseudomonas aeruginosa are devoid of mannuronan C-5 epimerase

Mucoid strains of Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas syringae var glycinia synthesize alginate, an extracellular copolymer comprising D-mannuronosyl and L-guluronosyl moieties. Extracellular mannuronan C-5 epimerase, which converts polymannuronate to alginate, was demonstrated in supernatant fluid from cultures of A. vinelandii. However, the enzyme could not be demonstrated, using the same assay, in supernatant fluids of cultures of mucoid strains of P. aeruginosa or of P. syringae var glycinia, or in cell-free sonic extracts of P. aeruginosa. The results suggest that the pathways of alginate biosynthesis in A. vinelandii and Pseudomonas species may differ.     

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.     

Biosynthesis of dermatan sulphate. Assay and properties of the uronosyl C-5 epimerase.

During biosynthesis of dermatan sulphate D-glucuronate (GlcA) residues are converted to L-iduronate (IdoA) residues via the reaction [Formula: see text]. The reaction occurs on the polymer level and is catalysed by a C-5 uronosyl epimerase. The reversible release of the C-5 hydrogen was utilized as a measure of the enzyme activity with 5-3H-labelled chondroitin as a substrate. 3H released during incubation was distilled and quantified by liquid-scintillation counting. The epimerase has a low pH optimum (5.6) and requires divalent cations, Mn2+ being the most efficient for activity. The Km for chondroitin is 1.2 x 10(-4) M. The epimerase is largely associated with the microsomal fractions (90%). Two-thirds of the activity can be solubilized by detergents. Microsomes from cultured fibroblasts contain two different uronosyl epimerases, one for the biosynthesis of heparan sulphate and one for that of dermatan sulphate. The two epimerases have different cofactor and pH requirements.     

Mutational analysis and characterization of nocardicin C-9' epimerase

The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806 was recently identified. Nocardicin A is the most potent of a series of monocyclic beta-lactam antibiotics produced by this organism. Its activity has been attributed to a syn-configured oxime moiety and a d-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework. Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5'-phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases. Insertional mutagenesis of nocJ abolished nocardicin A production, while the l-homoseryl isomer, isonocardicin A, was still observed. Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant. Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9' epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C. NocJ is the first member of a new class of beta-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.     

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.     

Comparative studies of NADP-malic enzyme from C-4 and C-3 plants

Some enzymological properties were studied comparatively for NADP-malic enzyme from various C₄- and C₃-plants. The enzyme from C₄-plants of “malate formers” showed relatively low Km(Mal) values (0.10 – 0.25 mM) and high pH optima (more than pH 7.4 – 7.8). Contractively, the enzyme from the other groups of higher plants including C₄-plants of “aspartate formers”, C₃-plants and CAM-plant showed relatively high Km(Mal) values (0.68 – 1.05 mM) and low pH optima (less than pH 7.4).     

Cloning and Expression of Three New Azotobacter vinelandii Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca²⁺-dependent polymer-level epimerization of β-d-mannuronic acid to α-l-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY. All three genes were sequenced and expressed in Escherichia coli. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5′-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5′ part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.Alginate is a linear copolymer composed of β-d-mannuronic acid (M) and its C-5 epimer, α-l-guluronic acid (G). The M and G residues are organized in blocks of consecutive M residues (M blocks), consecutive G residues (G blocks), or alternating M and G (MG blocks), and the lengths and distributions of the different block types vary among alginates isolated from brown algae or from different bacteria belonging to the genera Azotobacter and Pseudomonas (36, 37). Alginates are the most abundant polysaccharides in brown algae (comprising up to 40% of the dry matter), and their functions are to supply strength and flexibility to the algal tissues (38). The bacterium Azotobacter vinelandii produces alginate both as a vegetative state capsule and as an integrated part of a particular resting stage form (cyst) of this organism (31). The opportunistic pathogen Pseudomonas aeruginosa produces alginate as a capsule-like exopolysaccharide during infection of the lungs of cystic fibrosis patients (12, 23). Alginates from brown algae and A. vinelandii have M, G, and MG blocks (29, 36, 37), while alginates from P. aeruginosa and other Pseudomonas species do not contain G blocks (34, 36). In contrast to the alginates produced by brown algae, bacterial alginates are partially O-acetylated at O-2 and/or O-3 on mannuronic acid residues (36).The relative amount and distribution of G residues determine most of the physicochemical properties of the polymer. Alginates with G blocks can form gels by reversible cross-linking with divalent cations such as Ca²⁺, Ba²⁺, and Sr²⁺ (41), and the gelling and viscosifying properties of alginate are utilized in pharmaceutical, food, textile, and paper industries (26). In addition, alginate has a very interesting potential in a variety of biotechnological applications and in biomedicine. Alginate rich in M blocks stimulates cytokine production (27) and has a much higher antitumor activity than alginates with a high fraction of G blocks (14). G-rich alginates can be used for encapsulation of cells and enzymes (35), and Langerhans islets immobilized in alginates rich in G have been evaluated as a potential treatment for type 1 diabetes (39, 40).Both in brown algae and in alginate-producing bacteria, the polymer is first synthesized as mannuronan, and the enzyme mannuronan C-5-epimerase catalyzes the epimerization of M to G at the polymer level (7, 12, 21, 22). Ertesvåg et al. (7) have previously reported the cloning and expression of five genes encoding a family of Ca²⁺-dependent epimerases in A. vinelandii (algE1 to -5). The deduced AlgE protein sequences consist of two types of structural modules, designated A (385 amino acids each; one or two copies) and R (155 amino acids each; one to seven copies), and each R module contains four to six nine-amino-acid-long repeated sequences corresponding to putative Ca²⁺-binding motifs. The molecular masses of AlgE1 to -5 vary from 57.7 (AlgE4) to 191 kDa (AlgE3), depending on the number of A and R modules in the proteins. Four of the epimerase genes are clustered in the chromosome (algE1 to -4), while algE5 is located in another part of the A. vinelandii genome. Nuclear magnetic resonance (NMR) spectroscopy analyses demonstrate that the reaction products at least of AlgE2 and AlgE4 differ with respect to sequence distributions of M and G residues. AlgE2 leads to formation of mainly G blocks, while AlgE4 forms predominantly alginates with MG blocks.The A. vinelandii chromosome also encodes a Ca²⁺-independent mannuronan C-5-epimerase, designated AlgG (30). Sequence alignments demonstrate that algG does not belong to the algE gene family but shares 66% sequence identity to a mannuronan C-5-epimerase gene (also designated algG) from P. aeruginosa (12). The algG gene in P. aeruginosa is localized in a cluster of alg genes encoding enzymes involved in alginate biosynthesis, and sequence analysis of genomic DNA flanking algG in A. vinelandii suggests that this gene also is part of an alg gene cluster organized as in P. aeruginosa (30).Southern blot analysis of genomic A. vinelandii DNA using the 5′-terminal 800 bp in the A sequence of algE2 as the probe (A probe) demonstrated that the chromosome probably encodes more A-like sequences than are present in algE1 to -5 (7). In this report, we show that the A. vinelandii genome encodes two additional mannuronan C-5-epimerase genes, designated algE6 and algE7, and also a third highly related gene apparently not encoding an active epimerase.