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Polysaccharide epimerases

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Epimerases and racemases catalyse the inversion of stereochemistry in substrate molecules. Epimerases invert the stereochemistry of only one stereogenic center in a molecule that has at least two, whereas racemases catalyze the racemization of pure enantiomers, usually through the inversion of a stereogenic center in a molecule that possesses just one. These enzymes allow organisms to utilize several different stereoisomers of the same compounds. Whereas most enzymes catalyze reactions where reactants and products have only one stereochemistry, epimerases and racemases must be able to accommodate two different stereoisomers in their active site. Many epimerases and racemases act by breaking an acidic C-H bond next to a carbonyl functional group to give an enol or enolate, and then reform it with the new stereochemistry [1], while others break a C-H bond of an alcohol, acting through an oxidation/reduction sequence.

van Overtveldt et al. classified 39 known carbohydrate epimerase specificities into 14 different families based on sequence alignments [2]. Five different epimerization mechanisms were described: I) deprotonation/reprotonation, II) transient keto intermediate, III) carbon-carbon bond cleavage, IV) nucleotide elimination, and V) mutarotation. Members of a family share mechanism and conserved catalytic residues. Only two of the 14 families are active on polysaccharides: CEP7 containing mannuronan C5-epimerases (alginate epimerases) and CEP8 containing heparosan-N-sulfate-glucuronate 5-epimerase (heparosan epimerase). Together with the unclassified dermatan sulfate C5-epimerase (chondroitin epimerase), these enzymes perform a similar 'polymer-level' C5-epimerization on polysaccharides consisting of hexuronic acids. Alginates and the glycosaminoglycans heparin/heparan and dermatan sulfate are block copolymers of the two monomers created by these epimerases. In alginates, the epimerization is from d-mannuronic acid to l-guluronic acid, while in the glycosaminoglycans it is from d-glucuronic acid to l-iduronic acid [3, 4, 5, 6].

Alginate and glucosaminoglycan epimerases utilize a deprotonation/reprotonation mechanism where neutralization of the sugar carboxyl group is followed by proton abstraction from one side of the sugar ring, creating a resonance stabilized enol or enolate intermediate. The pKa value of the hydrogen is lowered due to this resonance stabilization [1, 6]. For the mannuronan C5-epimerase, the intermediate is thought to be an enolate anion, while for the heparin epimerase it could be a neutral enol [7]. For dermatan epimerases, it is proposed that deprotonation at C5 leads to elimination of the sugar attached at C4, with cleavage of the bond to the glycosidic oxygen, affording a 4,5-unsaturated intermediate that undergoes an addition reaction to reform the glycoside but with the proton added from the opposite side to give the epimer [8]; this elimination/addition mechanism has also been proposed as an alternative for alginate epimerases [9]. The mechanism is probably similar to that of Polysaccharide Lyases, which undergo a β-elimination to afford a 4,5-unsaturated hexenuronic acid product [10].

Despite performing similar reactions, the three hexuronyl C5-epimerase families share almost no sequence or structural similarity [2, 11].

Mannuronan C5-epimerases

Substrate specificity

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 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 [3, 12, 13]. Alginate is an anionic polysaccharide made by brown seaweeds, some species of red algae, and bacteria of the gram-negative genera Pseudomonas and Azotobacter [14, 15, 16, 17, 18]. The functions of alginate in different organisms relate to structure, protection, and surface adhesion [19, 20, 21, 22]. Alginate is a copolymer of the two 1,4-linked epimers [23, 24, 25], and by changing the relative amounts of the M and G monomers the epimerases fine-tune the properties of alginate [26]. Initially, 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 [13, 27, 28]. This epimerization is not random; it creates block structures of M, G, or alternating MG [29, 30], 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 [31, 32].

Mannuronan C5-epimerases exist both in algae and in bacteria [3, 33]. Gene analyses propose as many as 31 different genes encoding putative mannuronan C-5 epimerases in the brown algae Ectocarpus [34]. However, the algal epimerases are difficult to express and as a result the bacterial enzymes have been studied most extensively [34, 35]. Two categories of bacterial mannuronan C5-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 spp. are only known to produce AlgG [28, 36, 37], while A. vinelandii contains seven active AlgE enzymes, AlgE1-7, in addition to AlgG [38, 39, 40, 41]. Recently, three AlgE enzymes were characterized in A. chroococcum and denoted AcAlgE1-3 [42]. A mutant strain of P. fluorescens without the algG gene creates pure mannuronan [43]. This strain can be used to produce an unepimerized substrate, which is useful for the study of the epimerization reaction. Methods for studying this are discussed below.

Product profiles

The assorted epimerases of A. vinelandii give slightly different product profiles that makes it possible for the bacterium to tailor its alginate structure to fulfill different functions [39, 44]. This is done in three different ways. Firstly, some AlgE enzymes can only create MG-blocks, while others also create G-blocks. Secondly, different epimerases create block stretches of different lengths. Lastly, one of the seven A. vinelandii AlgE epimerases (AlgE7) has a dual epimerase/lyase activity and thus modifies the polymer length [41]. Two of the three AlgE enzymes found in A. chroococcum are homologous to AlgE7 and display the same bifunctional activity [42, 45]. Weak lyase activity has been observed in other AlgEs, although it is not certain whether this serves a function or if it is the result of failed epimerization reactions [46, 47].

Catalytic reaction

The extracellular A. vinelandii AlgE enzymes consist of different combinations of a catalytic module, the A-module, and a smaller R-module that is thought to modify binding [44, 48]. The enzymes' direction of movement along the substrate is unknown, but there are indications that they move along their polymeric substrate from the non-reducing to the reducing end [47, 49]. The epimerases show various degrees of processivity [47, 50, 51, 52], with AlgE4 catalyzing around 10-12 epimerizations before disassociating from the substrate [49, 53]. 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 [47, 49]. The reason why some epimerases can not form G-blocks may be related to their interactions with poly-MG sequences [53].


Figure 2. Three-step epimerase/lyase mechanism as proposed by Gacesa [10]. AA1, AA2 and AA3 denotes the amino acids, or potentially water molecules, responsible for each step. After neutralization of the negative charge on the D-mannuronate carboxyl group by AA1, AA2 can abstract the proton bound to C5. This results in an enolate intermediate that is resonance stabilized. A proton is subsequently donated to different groups depending on whether it is an epimerase or lyase reaction. In lyases, cleavage of the glycosidic bond forms an unsaturated residue denoted Δ and a proton is donated to the leaving group. In epimerases AA3 donates a proton to the C5 carbanion on the opposite side of the ring from the one that was abstracted, forming L-guluronate.

The proposed alginate epimerase mechanism is initiated with neutralization of the negative charge of the carboxylate group by either protonation or interaction with a positively charged amino acid (Figure 2) [10]. This is followed by abstraction of H-5 by a general base residue to form an enol or enolate, followed by protonation from the opposite side of the sugar ring by a general acid residue. The conformation of the monomer flips from 4C1 to 1C4 and changes it from β-d-mannuronate to α-l-guluronate. The chemical mechanism used by alginate epimerases is believed to be similar to the mechanism of Polysaccharide Lyases. This is supported by several of the A. vinelandii enzymes having both lyase and epimerase activity [41, 46, 47, 54]. 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. The NNHSY sequence is a common motif in both epimerases and lyases, and is believed to be important for catalysis or binding [43, 55, 56].

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.

The Dische carbazole reaction [57] can be used to measure both initial activity and endpoint conversion [13, 58, 59]. In this method, an increase in color intensity from mannuronic to guluronic acid is used to quantify the degree of epimerization.

Epimerization activity on 5-3H-alginate can be measured by observing tritium released into the solvent [60, 61]. This method has increased accuracy compared to the carbazole method is better suited for determination of kinetic parameters. Notably, because the substrate changes as epimerization occurs, classical Michaelis-Menten kinetics do not apply; instead, apparent values for Vmax and kcat are obtained. For AlgE4 they were determined to be 14.8 μmol min-1 mg-1 protein and 14 s-1, respectively [50].

A fast and sensitive method that does not require tritiated alginate involves the direct detection of the unsaturated product of the alginate lyase reaction, Δ, which absorbs at 230 nm [62]. This can be used to measure lyase activity directly [63], but it can also be used to measure epimerization in a coupled assay. This is done by treating epimerized alginate with an alginate lyase, e.g., AlyA from Klebsiella pneumoniae, which specifically cleaves at G-M and G-G linkages [64]. 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 can be assessed by acid hydrolysis of alginate [3, 29, 30]. Alternating blocks are readily hydrolysed and appear in the soluble fraction, while homopolymeric blocks are less reactive and remain insoluble. Subsequently, dissolution of the insoluble part followed by acid precipitation of G-blocks, allows the relative amounts of the three block structures to be roughly estimated. A more precise method uses either 13C-NMR [65, 66] or the more sensitive 1H-NMR [67, 68] spectroscopy to calculate block composition of alginate. This is done by measuring relative amounts of monomer dyads and triads.

Both 13C- and 1H-NMR spectroscopy have been used to monitor rates of epimerization, allowing measurement of kinetics and observation of the mode of action [54, 69, 70, 71].

Circular dichroism can distinguish between M and G [72, 73], and can be used to measure epimerase activity [74].

To determine 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 [75]. Size exclusion chromatography (SEC) is used to separate the products and the resulting block fractions are analyzed with high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) [76, 77], NMR spectroscopy, or SEC with multiple angle light scattering (SEC-MALS) [78].

Catalytic residues

Figure 3. Electrostatic surface potential and catalytic sites of A. AlgE4 (PDB ID 2PYH, [79]) and B. AlgG (PDB ID 4NK6, [80]). The maps were created using the APBS electrostatics plug-in [81] in PyMOL [82]. 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 structure of AlgE4 is shown as ball-and-stick with carbon atoms in green and oxygen atoms in red.

All polysaccharide 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 [35, 54, 79, 83] (See Figure 3A). The exact role of each residue in the mechanism is unclear. It has been suggested that tyrosine acts as the base in the reaction, marked AA2 in Figure 2, while the histidine is the acid, AA3 [79]. The two acidic residues might be important in maintaining the pKa values of amino acids in the active site, as well as orienting the catalytic base. In AlgG (Figure 3B), H319 is hypothesized to be the base, water to be the acid, and R345 to 'neutralize' the carboxyl group [80].

Role of calcium

The seven secreted epimerases of A. vinelandii, AlgE1-AlgE7, are calcium-dependent [3, 44, 84]. Activity increases with increasing calcium concentration up to around 5 mM calcium, with the optimum depending on the enzyme [13, 46, 50, 54, 85]. At the same time, increased calcium concentration gives a decrease in end-point conversion due to gelation of the product [13]. It is unknown whether calcium is important for the mechanism itself or if its role is to stabilize the structure (see three-dimensional structures below). Early studies indicated a role in enzyme stability, as the temperature stability increased at higher calcium concentrations [13]. In the 3D crystal structure of AlgE4's A-module, a calcium ion is bound in a hairpin loop away from the active site [79] (PDB ID 2PYH). Without the calcium ion, the loop would likely change conformation, which could destabilize the active fold of the enzyme. Calcium may also be involved in neutralizing the negative charge of the carboxylate anion, which is observed in the structurally similar alginate lyases from the polysaccharide lyase family 6 [86, 87, 88, 89]. The periplasmic AlgG epimerases do not require calcium for activity [28, 40].

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 [48, 90] (see Figure 3 for electrostatic surface potential maps). Assuming a monomer length of about 0.5 nm [53] and a binding groove length of about 5 nm [79], the catalytically active A-module can accommodate around 10 sugar residues. The R-module seems to be able to bind around 5 alginate monomers [48]. The epimerases probably require different substrate lengths to be able to initiate epimerization. For instance, AlgE4 seems to need at least a hexamer [49], while AlgE6 and AlgE1 need eight to ten monomers [47].

Different epimerases create different block lengths: the larger epimerase AlgE1 appears to create longer blocks than AlgE6, which in turn seems to create longer blocks than AlgE4 [47, 49]. This could be due to differences in processivity. Since the alginate epimerases are processive, high affinity substrate binding may decrease their activity. The R-modules may have a role in processivity: even though the R-module from AlgE4 increases its binding compared to the A-module alone, R-modules from AlgE6 cannot individually tobind alginate [48]. The R-modules probably modulate binding and processivity in complex ways, not solely by increasing the binding affinity [44, 48, 53, 70, 90, 91].

Residue 307 has been highlighted as important for epimerase substrate specificity. It is located in a large loop in the A-module of the AlgE enzymes. In G-block formers, this residue is tyrosine, whereas in MG-block formers it is a phenylalanine [70]. Mutations of this residue can change the epimerization pattern [71]. Because this residue is located relatively distant from the active site it it suggests that substrate binding, and not the catalytically active residues, that gives rise to different product profiles for different alginate epimerases.

Three-dimensional structures

Figure 4. A. Cartoon representations of the A-module of AlgG (left) and AlgE4 (right). The three β-sheets are called PB1 (teal), PB2 (olive) and PB3 (maroon) according to nomenclature of β-helix folds [92]. A bound mannuronan trimer is shown in sticks in the binding groove of AlgE4. A calcium ion is coordinated close to the N-terminal, shown as an orange sphere. The N-terminal alpha helix capping the hydrophobic interior is shown in yellow. In AlgG, this is more complex and consists of two β-sheets in addition to the helix. B. The same structure as in A but seen from the C-terminal end. T1, T2 and T3 denotes the turns between the three sheets. The shape of the protein from this angle illustrates why the beta-helix is also called an L-solenoid. C. The C-terminal R-module of AlgE4. Five calcium ions are coordinated by the structure, shown as orange spheres.

The first 3D crystal structure of a mannuronan C5-epimerase was that of the AlgE4 A-module from A. vinelandii, which was solved at 2.1 Å resolution [79]. 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 second 3D crystal structure reported was of the P. aeruginosa non-modular AlgG epimerase, at 2.1 Å resolution [80] (Figure 4A). It is structurally similar to AlgE4's A-module. In 2016 the A-module of AlgE6 was deposited in the 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 [41]. The three structures are all around 70 Å long. R-modules of AlgE4 [90] (PDB ID 2AGM) and AlgE6 [48] (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 LXGGAGXDXn, a circular permutation of the motif GGXGXDX(L/I/F)X first found in RTX (repeats in toxins) toxins from Gram-negative bacteria [93, 94]. The motifs stabilize the fold by binding calcium ions tightly [95], and the R-modules of AlgE contains four to seven of them [26, 90]. 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 [38, 90, 96].

No structures have been reported for a complete multimodular epimerase. From low resolution SAXS-measurements of AlgE4 and AlgE6, the enzymes appear elongated, with the R-modules extending the binding grooves of the A-modules [48]. 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 [48].

The parallel β-helix fold found in the active modules of the epimerases was first encountered in another enzyme active on a polyanionic substrate, namely the pectate lyase C of Erwinia chrysanthemi [97]. It is also called a β-solenoid-type fold as it consists of repeated β strands, and it is a type L β-solenoid [98]. In the epimerases, the β-solenoid is found together with an N-terminal α-helix cap that shields the hydrophobic interior of the β-helix from the solvent. This combination is found in several other enzyme families of glycoside hydrolases (families GH28, GH49, GH55, GH82, and GH87 [99]) and of polysaccharide lyases (families PL1, PL3, PL6, and PL9 [100]): alginate lyases [88], dextranases [101], rhamnogalacturonases [102], polygalacturonase [103], β-1,3-glucanases [104], ι-carrageenases [105], endorhamnosidases [106], pectate and pectin lyases [97, 107, 108, 109] and chondroitin B (dermatan sulfate) lyases [86]. Since these enzymes have a high degree of structural similarity, it is thought that at least some of them diverged from a common ancestor [110], although convergent evolution is not unlikely [98].

Heparosan-N-sulfate-glucuronate 5-epimerase

Substrate specificities

Figure 5. Hexamer illustrating the most common monomers found in heparan sulfate. GlcNAc represents α-d-N-acetyl glucosamine, GlcA is β-d-glucuronate, GlcNS is 2-sulfamido-α-d-glucosamine, IdoA(2S) is 2-O-sulfo-α-l-iduronate, GlcNS(6S) is 2-sulfamido-α-d-glucosamine-6-O-sulfate and IdoA is α-l-iduronate. Glucosamine sulfated at 3-O (GlcNS(3S,6S)) and glucosamine with a free amine group (GlcN) also exist, but these are rarer and are not shown in the structure.

Heparin/heparan epimerase catalyzes epimerization of β-d-glucuronic acid (GlcA) to α-l-iduronic acid (IdoA) at the polymer level in the glycosaminoglycan heparan sulfate [111]. Heparan sulfate consists of long chains of a repeating disaccharide motif, 1,4-linked alternating monomers of GlcA or IdoA and α-d-glucosamine (GlcN). GlcN monomers can be N-acetylated or N-sulfated, and all three sugar units can be O-sulfated. While GlcA adopts a 4C1 chair conformation, IdoA exists in an equilibrium between 1C4 and the skew-boat conformation 2S0 [6]. Domains in heparan sulfate have various compositions of monomers and modifications, and long domains with high amounts of N-sulfated disaccharide units are called heparin [112]. Figure 5 shows a hexamer with common heparan sulfate monomers.

Heparan sulfate is found in all animal cells as proteoglycans. Due to its negative charge and diverse sequence motifs, the polysaccharide interacts with many different proteins and affect most cellular processes mainly through electrostatic interactions. Heparin is stored in secretory granules in mast cells and released when tissue is injured. When it binds to antithrombin it aids in inhibiting blood coagulation. Heparin is produced industrially as an anticoagulant; its roles in tissue injury and repair in cells are diverse and complex [6, 113].

The activity of the heparin/heparan epimerase requires that the GlcA/IdoA residue to be epimerised has a deacetylated and N-sulfated glucosamine linked to its C-4 (subsite -1). O-Sulfation of the residue to be epimerized, or of neighboring residues, inhibits epimerization. The residue linked to C-1 of the sugar unit in the active site can be N-sulfated or N-acetylated, but N-sulfation yields a better substrate. An octasaccharide is the minimal substrate size [114, 115].

The enzymatic reaction is reversible when the substrate has a glucosamine or sulfated glucosamine three residues away from the epimerization site towards the nonreducing end, or if this site is unoccupied. Conversely, if the substrate has an acetylated glucosamine in the same site the enzyme irreversibly introduces l-iduronic acid residues to the heparin chains. Because IdoA residues are substrates for sulfation reaction in vivo, and the resulting O-sulfated IdoA is not a substrate, epimerization is effectively irreversible in vivo [116, 117, 118].

An enzyme with a different substrate specificity has been identified from the giant African snail Achatina fulica [119]. The glycosaminoglycan acharan sulfate contains repeating units of N-acetylated glucosamine and O-sulfated IdoA. The epimerase creating IdoA in this polymer is named heparosan-glucuronate 5-epimerase since it is active on heparosan, as opposed to the heparosan-N-sulfate-glucuronate 5-epimerase that requires a deacetylated and sulfated substrate.

Catalytic reaction and mechanism

The catalytic reaction is thought to follow a similar mechanism to the mannuronan C5-epimerases described above, and also to that of heparin and dermatan sulfate lyases [120, 121]. A tyrosine acts as the proton acceptor and a glutamate as the proton donor. Based on structural analysis of substrate and product complexes the reaction is believed to proceed through a neutral enol intermediate, with ring distortion to facilitate epimerization [7]. Originally the intermediate was proposed to be an α-carbanion/enolate [122], as for the other polysaccharide epimerases [11]. Two other tyrosines are essential for activity, and they are hypothesized to be part of proton relay pathways between the active site and the solvent [7, 11]. Protonation of the enol is the rate-limiting step [7, 123]. Heparin/heparan epimerases do not require divalent cations for activity, unlike the alginate and dermatan sulfate epimerases [124]. However, one calcium ion is bound to each subunit in the crystal structure of human heparin/heparan epimerase [7]. In the crystal structure of zebrafish heparin/heparan epimerase, a water molecule is bound in the same position [11].

Three-dimensional structures

Figure 6. Cartoon representation of the dimeric assembly of human glucuronyl C5-epimerase (PDB ID 6HZZ) [7]. The N-terminal β-hairpin domain is colored in purple, the β-sandwich domain is colored in red and the (α/α)4-barrel domain is colored in green. One calcium ion is coordinated in the sandwich domain of each subunit and shown as an orange sphere. Three N-glycans bound to each subunit are shown in sticks and spheres.

The first crystal structure was reported in 2015, d-glucuronyl C5-epimerase from zebrafish (PDB ID 4PW2) [11]. The protein crystallizes as a homodimer and has three domains: an α-helical transmembrane region, a β-barrel domain, and a flexible N-terminal loop. An enzyme complex with an inhibiting heparin hexamer was also reported that defined the active site cleft (PDB ID 4PXQ).

The structure of human d-glucuronyl C5-epimerase has been reported (PDB ID 6HZZ) [7]. Like the zebrafish enzyme, it is a dimer where each subunit has three domains. The N-terminal domain consists of two antiparallel β-hairpins connected by a helix. This is followed by a β-sandwich domain, and a C-terminal (α/α)4-barrel domain containing the active site. The dimer is probably the active form of the enzyme. Figure 6 shows the dimeric structure of human glucuronyl C5-epimerase. Structures of an inactive mutant bound to a substrate and a product were also deposited (PDB IDs 6I01 and 6I02) [7].

Dermatan sulfate C5-epimerase

Substrate specificities

Figure 7. Heptamer motif of a dermatan sulfate chain showing the possible monomers. GalNAc(4S,6S) represents β-d-N-acetyl galactosamine 4,6-O-sulfate, GlcA is β-d-glucuronate, GalNAc is β-d-N-acetyl galactosamine, IdoA is α-l-iduronate, GalNAc(4S) is β-d-N-acetyl galactosamine 4-O-sulfate, IdoA(2S) is 2-O-sulfo-α-l-iduronate and GalNAc(6S) is β-d-N-acetyl galactosamine 6-O-sulfate.

Dermatan sulfate epimerases catalyze the epimerization of β-d-glucuronic acid (GlcA) to α-l-iduronic acid (IdoA) in polymers of glycosaminoglycans [125, 126, 127], an activity similar to heparin epimerase. The repeating disaccharide unit of dermatan sulfate is 1,3-linked GlcA or IdoA and 1,4-linked β-d-N-acetyl galactosamine (GalNAc). Sulfation can occur at C-2 of iduronic acid and C-4 and/or C-6 of galactosamine. Figure 7 illustrates the seven possible monomers after sulfation and epimerization, three for the hexuronates and four for galactosamine. Dermatan sulfate can have various lengths, degrees of epimerization, and sulfation patterns, and can bind to a variety of proteins [128]. The unepimerized polymer is called chondroitin sulfate. Although dermatan sulfate was earlier known as chondroitin B it is no longer classified as a chondroitin sulfate [129]. Chondroitin/dermatan sulfate exists as proteoglycans in extracellular matrixes in mammalian tissues, especially skin. The polysaccharide is involved in many different cell processes due to its sequence variability and diverse protein partners [128].

Dermatan sulfate consists of block structures of (IdoA-GalNAc)n, (GlcA-GalNAc)n, and hybrid domains containing both uronic acids. Two epimerases are known: dermatan sulfate epimerases 1 and 2, which have slightly different product patterns and which could be important in regulating dermatan sulfate domain formation. They are found in a variety of animals, including humans [127].

Dermatan is a better substrate for the epimerases than chondroitin, and the epimerases are inactive on sulfated substrates [130]. However, the co-incubation of epimerases and sulfotransferases synergistically promotes the formation of iduronic acid. The epimerases physically interact with the sulfotransferase and processively form long iduronic acid-containing domains [5, 131]. The optimal minimal substrate is an octamer [132].

Catalytic reaction and mechanism

A mechanism is suggested where a proton is abstracted from one side of glucuronic acid by a histidine, the glycosidic bond is cleaved by the involvement of a tyrosine and another histidine attaching a proton to the other side of the ring. This will involve a 4,5-unsaturated intermediate, similar to the end product of polysaccharide lyases. The glycosidic bond would be formed again by the tyrosinate ion or a second general acid [8]. The two histidines could switch roles as acid and base, giving rise to the reversibility seen in vitro [133]. The enzyme requires divalent cations, preferably Mn2+, and N-glycosylation for activity [8, 125]. It processively epimerizes four to five monomers from the reducing towards the non-reducing end, with decreased binding affinity for each processive step [132]. In vivo, the concomitant action of epimerase and sulfotransferase could account for a higher processivity, as well as the irreversible formation of iduronic acid [130, 131, 134].

Three-dimensional structures

The crystal structure of human dermatan sulfate epimerase 1 was deposited in PDB in 2020 (PDB ID 6HZN). It contains an (α/α)6 toroid N-terminal domain, a β-sheet domain, and a C-terminal domain with a long α-helix. A manganese ion is bound close to the putative active site, which is located in a groove between the two main domains.


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