Polysaccharide epimerases

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• Responsible Curator: ^^^Finn Aachmann^^^

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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], see Figure 1. 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].

Figure 1. 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 Figure 2 [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

Several methods have been used either to measure catalytic rates, or to characterize the epimerized product in terms of relative amounts of M, G and block compositions at different conditions and treatment times.

The Dische carbazole reaction [46] was used in the 1970s to measure both initial activity and end point conversion [3, 47, 48]. In this method an increase in color intensity from mannuronic to guluronic acid is used to quantify the degree of epimerization.

In the 1980s, epimerization activity on 5-³H-alginate was measured by observing tritium released into the solvent [49, 50]. This method had an increased accuracy compared to the carbazole method and was more suited to determine kinetic constants. Although the substrate changes during epimerization so classical Michaelis-Menten kinetics cannot be applied, apparent values for V_max and k_cat for AlgE4 were determined to be 14.8 μmol min^-1 mg^-1 protein and 14 s^-1, respectively [38].

Around the same time, another fast and sensitive method that did not require tritiated alginate was established [51]. The non-saturated product of alginate lyase reactions, Δ, has absorbance at 230 nm. This can be used to measure lyase activity directly [52], but it can also be used to measure epimerization indirectly. This is done by treating epimerized alginate with an alginate lyase, e.g., AlyA from Klebsiella pneumoniae that specifically cleaves at G-M and G-G linkages [53]. Formation of Δ, monitored by measuring absorbance at 230 nm, is then assumed to be directly proportional to the amount of G produced by the epimerase. Block composition was initially measured by acid hydrolysis of alginate [1, 19, 20]. Alternating blocks are readily hydrolysed and appear in the soluble fraction, while homopolymeric blocks are more protected and remain insoluble. By subsequently dissolving the insoluble part followed by acid precipitation of G-blocks, relative amounts of the three block structures was roughly determined. A much more precise method was later established, using either ¹³C-NMR [54, 55] or the more sensitive ¹H-NMR [56, 57] to calculate block composition of alginate. This is done by measuring relative amounts of monomer dyads and triads. These are important methods for characterization of alginate today.

Both ¹³C- and ¹H-NMR have been used to follow the epimerization reaction over time, making it possible to observe both kinetics and mode of action [43, 58, 59, 60].

Another method that can distinguish between M and G is circular dichroism [61, 62], and this has also been used to measure epimerase activity [63].

To get knowledge about block length and distribution, lyases with four different specificities (cutting the alginate chain at either M-M, G-G, M or G) can be used [64]. Size Exclusion Chromatography (SEC) can then separate the products, before the resulting block fractions can be analyzed with High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) [65, 66], NMR or SEC with multiangle laser light scattering (SEC-MALLS) [67].

Catalytic residues

All known epimerases share a YG(F/I)DPH(D/E) motif located in the +1 subsite. In the AlgE epimerases the catalytic residues are identified as the four essential amino acids Y149, D152 and H154 of this motif, in addition to D178 that is lacking in AlgG [25, 43, 68, 69] (See Figure 3A). A lot is still unclear about the catalytic mechanism and of the exact role of each catalytic residue. It has been suggested that tyrosine acts as the catalytic base in the reaction, marked AA2 in Figure 3, while the histidine is the catalytic acid, AA3 [69]. The two acidic residues might be important in maintaining the pKa of this site, as well as orienting the catalytic base. In AlgG (Figure 3B), H319 is hypothesized to be the catalytic base, water to be the catalytic acid and R345 to neutralize the carboxyl group [70].

Figure 3. Electrostatic surface potential and catalytic sites of A. AlgE4 (PDB ID 2PYH, [69]) and B. AlgG (PDB-ID 4NK6, [70]). The maps are created using the APBS electrostatics plug-in [71] in PyMOL [72]. The scale is from -5 to +5 kT/e, where negative potential is colored red and positive potential is colored blue. The mannuronate trimer in the crystal strucutre of AlgE4 is shown as ball-and-stick with carbon atoms in green and oxygen atoms in red.

Role of calcium

The seven secreted epimerases of A. vinelandii, AlgE1-AlgE7, are all calcium-dependent [1, 33, 73]. Activity increases with increasing calcium concentration up to around 5 mM calcium, with the optimum depending on the enzyme [3, 34, 38, 43, 74]. At the same time increased calcium concentration gives a decrease in end point conversion due to gelation of product [3]. It is unknown whether calcium is important for the mechanism itself or if its only role is to stabilize the structure (see next section). Early studies indicated it had a role in enzyme stability, as the temperature stability increased at higher calcium concentrations [3]. In the resolved crystal structure of AlgE4's A-module, a calcium ion is bound in a hairpin loop away from the active site [69] (PDB-ID 2PYH). Without the calcium ion the loop would likely change conformation, which could destabilize the active fold of the enzyme. If calcium has an additional role in the catalytic activity, it could be in neutralizing the negative charge of the carboxylic anion. This is observed in the structurally similar alginate lyases from the polysaccharide lyase family 6 [75, 76, 77, 78]. The periplasmic AlgG epimerases do not require calcium for their activity [18, 30].

Substrate binding

Alginate is a polyelectrolyte, and substrate binding probably occurs mainly through electrostatic interactions. The A. vinelandii epimerases are modular, and together the modules form an extended binding groove lined by charged residues [36, 79] (see Figure 3 for electrostatic surface potential maps). Assuming a monomer length of about 0.5 nm [41] and a binding groove length of about 5 nm [69], the catalytically active A-module can accommodate around 10 sugar residues. The R-module seems to be able to bind around 5 alginate monomers [36]. The epimerases probably require different substrate lengths to be able to start epimerization. For instance, AlgE4 seems to need at least a hexamer [37], while AlgE6 and AlgE1 need eight to ten monomers [35].

They also create different block lengths: the larger epimerase AlgE1 appears to create longer blocks than AlgE6, which in turn seems to be create longer blocks than AlgE4 [35, 37]. This could be due to differences in processivity. Since the alginate epimerases are processive, a too strong substrate binding could actually decrease their activity. The R-modules could have a role here: even though the R-module from AlgE4 increases its binding compared to the A-module alone, R-modules from AlgE6 are not individually able to bind alginate [36]. The R-modules probably modulate binding and processivity in complex ways, not simply by increasing the binding strength [33, 36, 41, 59, 79, 80].

Another discovery related to substrate specificity is the importance of residue 307, located in a large loop in the A-module of the AlgE enzymes. In the G-block formers this residue is a tyrosine, whereas in the MG-block formers it is a phenylalanine [59]. Mutations of this residue have been shown to change the epimerization pattern accordingly [60]. As this residue is relatively far away from the active site it could be the substrate binding, not the catalytic mechanism, that gives rise to different product profiles.

Three-dimensional structures

In 2008 the structure of the AlgE4 A-module from A. vinelandii was solved at 2.1 Å resolution [69]. This represents the first crystal structure of a mannuronan C5-epimerase. It shows a right-handed parallel β-helix fold with an N-terminal α-helix cap and an extended binding groove, see Figure 4B. Protruding from the binding groove are three flexible loops, slightly enclosing the binding surface. One of the two molecules of the asymmetric unit has a mannuronan trimer bound in its binding groove. A calcium ion is coordinated in proximity to the active site, at the N-terminal end. The next crystal structure to be solved was of the P. aeruginosa non-modular AlgG epimerase, also at 2.1 Å resolution [70] (Figure 4A). It is structurally similar to AlgE4's A-module. In 2016 the A-module of AlgE6 was deposited in PDB (PDB ID 5LW3) at 1.19 Å resolution, and it is almost identical to AlgE4's A-module. These two A-modules also share the highest sequence homology of the AlgE A-modules [31]. The three structures are all around 70 Å long.

R-modules of AlgE4 [79] (PDB ID 2AGM) and AlgE6 [36] (PDB IDs 2ML1, 2ML2 and 2ML3) from A. vinelandii were solved by NMR. The R-modules have an overall ellipsoid or spherical shape, continuing the parallel β-sheets of the A-module with a parallel β-roll fold (See Figure 4C). A common feature of this fold is a repeated nonapeptide motif LXGGAGXDX$_n$, a circular permutation of the motif GGXGXDX(L/I/F)X first found in RTX (repeats in toxins) toxins from Gram-negative bacteria [81, 82]. The motifs stabilize the fold by binding calcium ions tightly [83], and the R-modules of AlgE contains four to seven of them [16, 79]. The core part of the R-modules is around 40 Å long. At the C-terminal of the last R-module of each enzyme is an unstructured region of about 20 amino acids. This is thought to function as a secretion signal for a transporter that secretes the enzymes out of the cell [28, 79, 84].

A structure of a complete modular epimerase is lacking. From SAXS-measurements of AlgE4 and AlgE6 the enzymes appear elongated, with the R-modules extending the binding grooves of the A-modules [36]. Some flexibility between the modules is observed, and NMR-studies indicate a flexible linker between the A- and the R-modules. Radii of gyration are calculated to be 31 Å for AlgE4 and from 52-55 Å for AlgE6. Maximum distances are around 100 Å for AlgE4 and around 180 Å for AlgE6 [36].

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