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Polysaccharide epimerases

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  • Author: ^^^Margrethe Gaardlos^^^ and ^^^Anne Tondervik^^^
  • Responsible Curator: ^^^Finn Aachmann^^^

Introduction

Classification

Mannuronan C5-epimerases

Substrate specificity

Mannuronan C5-epimerases are a group of enzymes that catalyze epimerization at the polymer-level of β-d-mannuronic acid residues (hereafter denoted M) into α-l-guluronic acid residues (hereafter denoted G) in alginate [1, 2, 3]. Alginate is an anionic polysaccharide made by brown seaweeds, some species of red algae, and the gram-negative bacterial genera Pseudomonas and Azotobacter [4, 5, 6, 7, 8]. The function of alginate in the different organisms are various, and related to structure, protection and surface adhesion [9, 10, 11, 12]. Alginate is a copolymer of the two 1-4 linked epimers [13, 14, 15], and by changing the composition of the two monomers the epimerases fine-tune the properties of the polymer [16].

At first, alginate is made as a homopolymer of M in the cell. Epimerases then convert some of the M residues in the polymer into G-residues [3, 17, 18]. This epimerization is not random and creates block structures of M, G or alternating MG [19, 20]. Alginate residues that are oxidized or acetylated are not substrates for the epimerases, and acetylation of alginate could be a way to control epimerization in nature [21, 22].

A. Haworth structures of d-mannuronic acid (M) and l-guluronic acid (G). B. The three different block structures found in alginate: non-epimerized M-blocks, MG-blocks and G-blocks.

Mannuronan C5-epimerases exist both in algae and in bacteria [1, 23]. Gene analyses propose as many as 31 different genes encoding putative mannuronan C-5 epimerases in the brown algae Ectocarpus [24]. However, the algal epimerases are difficult to express and it is the bacterial enzymes that have been studied most extensively [24, 25]. Two categories of bacterial mannuronan C-5-epimerases have been described: the periplasmic AlgG and the extracellular and calcium dependent AlgE. AlgG creates single G residues in stretches of mannuronan, while the AlgE enzymes are processive and create MG-blocks and G-blocks. Pseudomonas is only known to produce AlgG [18, 26, 27], while A. vinelandii contains seven active AlgE enzymes in addition to AlgG [28, 29, 30, 31]. A mutant strain of P. fluorescens without the algG gene creates pure mannuronan [32]. This strain can be used to produce unepimerized substrate, which is useful for the study of the epimerization reaction. Methods for studying this are discussed in a later section.

Product profiles

The abundance of epimerases giving slightly different product profiles in A. vinelandii makes it possible for the bacteria to tailor alginate so it can fulfill different functions [29, 33]. This is done in three different ways. Firstly, some AlgE enzymes are only capable of creating MG-blocks, while others also create G-blocks. Secondly, different epimerases create block stretches of different lengths. Lastly, one of the known AlgE epimerases has a dual epimerase/lyase activity and thus modifies the polymer length [31]. Weak lyase activity has also been observed in other AlgEs [34, 35]. It is not certain whether this serves a function or if it is the result of failed epimerization.

Catalytic reaction

The extracellular A. vinelandii AlgE enzymes are studied extensively. They consist of different combinations of an independently catalytic module, the A-module, and a smaller R-module thought to modify binding [33, 36]. The enzymes' direction of movement along the substrate is not determined, but there are indications that they move along their polymeric substrate from the non-reducing to the reducing end [35, 37]. The epimerases show various degrees of processivity [35, 38, 39, 40], where AlgE4 catalyzes around 10-12 epimerizations before disassociating from the substrate [37, 41]. Epimerases are thought to only epimerize every other residue in one binding event, which means that the G-block formers will need to bind the MG-product of the first reaction again to form G-blocks [35, 37]. The reason why some epimerases can not form G-blocks may be related to their interactions with poly-MG [41].

Mechanism

The mechanism is suggested to be similar to the lyase mechanism, as illustrated in the figure [42]. This is supported by several of the A. vinelandii enzymes having both lyase and epimerase activity [31, 34, 35, 43]. Different versions of NNHSY is a common motif in both epimerases and lyases, and it is implicated to be important for catalysis or binding [32, 44, 45].

The proposed epimerase mechanism is initiated with neutralization of the negative charge of the carboxylate group. This is followed by abstraction of H5 by a catalytic base and ends with an addition of another proton to the opposite side of the sugar ring by a catalytic acid. The conformation of the monomer flips from 4C1 to 1C4, and changes it from β-d-mannuronate to α-l-guluronate. In the lyase mechanism, the second step is a β-elimination of the 4-O-glycosidic bond to form a 4-deoxy-l-erythro-hex-4-enepyranosyluronate, called Δ, at the non-reducing end.


Methods to study the reaction

Catalytic residues

Role of calcium

Substrate binding

Three-dimensional structures

References

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