The Recombinant Azotobacter vinelandii Mannuronan C-5-Epimerase AlgE4 Epimerizes Alginate by a Nonrandom Attack Mechanism

Abstract

The Ca 2؉ -dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of ␤-D-mannuronic acid (M) to ␣-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 Ca 2؉ , and at temperatures near 37°C. Sr 2؉ was found to substitute reasonably well for Ca 2؉ in activation, whereas Zn 2؉ 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.

The Pseudomonas syringae Genome Encodes a Combined Mannuronan C-5-epimerase and O-Acetylhydrolase, Which Strongly Enhances the Predicted Gel-forming Properties of Alginates

“…The 1-13 C-labeled and unlabeled mannuronan (100% M, chemically deacetylated) were prepared from an epimerase-defective (algG) P. fluorescens mutant (18,43). Alginate containing alternating MG residues (MG-alginate) was produced in vitro from mannuronan by using recombinantly produced AlgE4 (23). Alginate from the leaves of Laminaria hyperborea and LF 10/60, which is an L. hyperborea stripe alginate, were obtained from FMC Biochemicals, Drammen, Norway.…”

“…Because both enzymes are identical in their N-terminal parts and are expressed in the same vector system, these results strongly indicated that the AlgE4 R-module can stimulate the activity of the PsmEA-module, despite the significant differences (61% identity) in the primary sequences of the A-modules of these two enzymes. Coomassie-stained SDS-PAGE gels and Western blots showed that an exact quantification of this stimulation was difficult to obtain due to the production of several forms of these enzymes in E. coli, as previously described for AlgE4 (23). The epimerization pattern generated by PsmEAR was also determined by 1 H NMR spectroscopy, using partially purified enzyme, as described for PsmE and PsmEA.…”

“…AlgE2), and MG block-forming (e.g. AlgE4) enzymes (22)(23)(24), which are composed of varying numbers of two modules, A (about 385 amino acids) and R (about 150 amino acids). By using AlgE1 as a model, the A module was shown to determine the epimerization pattern and to be sufficient for epimerization, whereas the reaction rate is influenced by the R modules (25).…”

Use of protein trans‐splicing to produce active and segmentally 2H, 15N labeled mannuronan C5‐epimerase AlgE4

“…The NMRbased assay, 23 whose result is shown in Figure 6, certified that A-[ 15 N]-R epimerizes poly-M-alginate to the typical MG-pattern characteristic for AlgE4. 24 As the A-module alone is also active, 30 this is not necessarily a proof for the presence of active AlgE4. However, the epimerisation rate of the A-module alone is significantly lower than that of full-size AlgE4.…”

Mannuronan C-5 Epimerases Suited for Tailoring of Specific Alginate Structures Obtained by High-Throughput Screening of an Epimerase Mutant Library

“…Although highly homologous, these enzymes each create characteristic M/G patterns producing alginates with different properties. AlgE4 which is the smallest epimerase containing one A-module and one R-module makes predominantly alternating M/G structure acting on polyM by a processive mode of action [14][15][16] . AlgE1, AlgE2, AlgE3, AlgE5 and AlgE6 make G-blocks of varying lengths, and AlgE6 is the epimerase able to make the longest G-block structures when acting on polyM 17 .…”