Polysaccharide epimerases - Revision history

Harry Brumer: Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

2021-12-18T21:16:18Z

Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

Margrethe Gaardlos at 20:10, 21 January 2021

2021-01-21T20:10:40Z

Spencer Williams: /* Substrate specificities */

2020-11-15T23:17:04Z

Substrate specificities

Spencer Williams: /* Substrate specificities */

2020-11-15T23:16:22Z

Substrate specificities

Spencer Williams: /* Catalytic residues */

2020-11-15T23:15:46Z

Catalytic residues

Spencer Williams: /* Mechanism */

2020-11-15T23:15:05Z

Mechanism

Spencer Williams: /* Substrate specificities */

2020-11-15T23:12:29Z

Substrate specificities

Spencer Williams: /* Three-dimensional structures */

2020-11-15T23:10:50Z

Three-dimensional structures

Spencer Williams: /* Catalytic reaction and mechanism */

2020-11-15T23:08:50Z

Catalytic reaction and mechanism

Spencer Williams: /* Substrate specificities */

2020-11-15T23:06:06Z

Substrate specificities

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 {{CuratorApproved}}
-* Author: ^^^Margrethe Gaardlos^^^ and ^^^Anne Tondervik^^^
+* Author: [[User:Margrethe Gaardlos|Margrethe Gaardlos]] and [[User:Anne Tondervik|Anne Tondervik]]
-* Responsible Curator:  ^^^Finn Aachmann^^^
+* Responsible Curator:  [[User:Finn Aachmann|Finn Aachmann]]
 ----
 == Introduction ==

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 === Catalytic residues ===
 [[Image:binding_actsite2.png|thumb|600px|Figure 3. Electrostatic surface potential and catalytic sites of A. AlgE4 ([{{PDBlink}}2PYH PDB ID 2PYH], <cite> Rozeboom2008 </cite>) and B. AlgG ([{{PDBlink}}4NK6 PDB ID 4NK6], <cite> wolfram2014 </cite>). The maps were created using the APBS electrostatics plug-in <cite> jurrus2018 </cite> in PyMOL <cite> PyMOL </cite>. 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 <cite> svanem2001, nyvall2003, Douthit2005, Rozeboom2008 </cite> (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 <cite> Rozeboom2008 </cite>. The two acidic residues might be important in maintaining the pK<sub>a</sub> 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 <cite> wolfram2014 </cite>.
+All known alginate 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 <cite> svanem2001, nyvall2003, Douthit2005, Rozeboom2008 </cite> (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 <cite> Rozeboom2008 </cite>. The two acidic residues might be important in maintaining the pK<sub>a</sub> 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 <cite> wolfram2014 </cite>.
 === Role of calcium ===

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 == Dermatan sulfate C5-epimerase ==
 === Substrate specificities ===
-[[Image:Dermatansulfate.png|thumb|600px|Figure 7. The figure shows a heptamer motif in a dermatan sulfate chain consisting of the possible monomers. GalNAc(4S,6S) represents &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 4,6-''O''-sulfate, GlcA is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronate, GalNAc is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine, IdoA is &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate, GalNAc(4S) is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 4-''O''-sulfate, IdoA(2S) is 2-''O''-sulfo-&alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate and GalNAc(6S) is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 6-''O''-sulfate.]]
+[[Image:Dermatansulfate.png|thumb|600px|Figure 7. Heptamer motif of a dermatan sulfate chain showing the possible monomers. GalNAc(4S,6S) represents &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 4,6-''O''-sulfate, GlcA is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronate, GalNAc is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine, IdoA is &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate, GalNAc(4S) is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 4-''O''-sulfate, IdoA(2S) is 2-''O''-sulfo-&alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate and GalNAc(6S) is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 6-''O''-sulfate.]]
 Dermatan sulfate epimerases catalyze the epimerization of &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronic acid (GlcA) to &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronic acid (IdoA) in polymers of glycosaminoglycans <cite>malmstrom1982, maccarana2006, pacheco2009b</cite>, an activity similar to heparin epimerase. The repeating disaccharide unit of dermatan sulfate is 1,3-linked GlcA or IdoA and 1,4-linked &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''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 <cite>trowbridge2002</cite>. 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 <cite>NLM2020</cite>. 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 <cite>trowbridge2002</cite>.

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 == Heparosan-N-sulfate-glucuronate 5-epimerase ==
 === Substrate specificities ===
-[[Image:Heparansulfate.png|thumb|600px|Figure 5. Hexamer motif illustrating the most common monomers found in heparan sulfate. GlcNAc represents &alpha;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl glucosamine, GlcA is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronate, GlcNS is 2-sulfamido-&alpha;-<font style="font-feature-settings: 'smcp'">d</font>-glucosamine, IdoA(2S) is 2-''O''-sulfo-&alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate, GlcNS(6S) is 2-sulfamido-&alpha;-<font style="font-feature-settings: 'smcp'">d</font>-glucosamine-6-''O''-sulfate and IdoA is &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-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.]]
+[[Image:Heparansulfate.png|thumb|600px|Figure 5. Hexamer illustrating the most common monomers found in heparan sulfate. GlcNAc represents &alpha;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl glucosamine, GlcA is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronate, GlcNS is 2-sulfamido-&alpha;-<font style="font-feature-settings: 'smcp'">d</font>-glucosamine, IdoA(2S) is 2-''O''-sulfo-&alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate, GlcNS(6S) is 2-sulfamido-&alpha;-<font style="font-feature-settings: 'smcp'">d</font>-glucosamine-6-''O''-sulfate and IdoA is &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-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 &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronic acid (GlcA) to &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronic acid (IdoA) at the polymer level in the glycosaminoglycan heparan sulfate <cite>hook1974</cite>. Heparan sulfate consists of long chains of a repeating disaccharide motif, 1,4-linked alternating monomers of GlcA or IdoA and &alpha;-<font style="font-feature-settings: 'smcp'">d</font>-glucosamine (GlcN). GlcN monomers can be ''N''-acetylated or ''N''-sulfated, and all three sugar units can be ''O''-sulfated. While GlcA adopts a <sup>4</sup>''C''<sub>1</sub> chair conformation, IdoA exists in an equilibrium between <sup>1</sup>''C''<sub>4</sub> and the skew-boat conformation <sup>2</sup>''S''<sub>0</sub>  <cite>valla2001</cite>. 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 <cite>gallagher1985</cite>. Figure 5 shows a hexamer with common heparan sulfate monomers.

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 === Catalytic residues ===
-[[Image:binding_actsite2.png|thumb|600px|Figure 3. Electrostatic surface potential and catalytic sites of A. AlgE4 ([{{PDBlink}}2PYH PDB ID 2PYH], <cite> Rozeboom2008 </cite>) and B. AlgG ([{{PDBlink}}4NK6 PDB ID 4NK6], <cite> wolfram2014 </cite>). The maps are created using the APBS electrostatics plug-in <cite> jurrus2018 </cite> in PyMOL <cite> PyMOL </cite>. 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.]]
+[[Image:binding_actsite2.png|thumb|600px|Figure 3. Electrostatic surface potential and catalytic sites of A. AlgE4 ([{{PDBlink}}2PYH PDB ID 2PYH], <cite> Rozeboom2008 </cite>) and B. AlgG ([{{PDBlink}}4NK6 PDB ID 4NK6], <cite> wolfram2014 </cite>). The maps were created using the APBS electrostatics plug-in <cite> jurrus2018 </cite> in PyMOL <cite> PyMOL </cite>. 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 <cite> svanem2001, nyvall2003, Douthit2005, Rozeboom2008 </cite> (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 <cite> Rozeboom2008 </cite>. The two acidic residues might be important in maintaining the pK<sub>a</sub> 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 <cite> wolfram2014 </cite>.

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 === Mechanism ===
-[[Image:Mech_epimerase_lyase.png|thumb|600px|Figure 2. The three-step mechanism as proposed by Gacesa <cite> gacesa1987 </cite>. AA1, AA2 and AA3 denotes the amino acids, or potentially also water molecules, responsible for each step. After neutralization of the negative charge on the mannuronate carboxyl group by AA1, AA2 can abstract the proton bound to C5. This results in resonance stabilization of the enolate anion intermediate. 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 &Delta; 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 guluronate.]]
+[[Image:Mech_epimerase_lyase.png|thumb|600px|Figure 2. Three-step epimerase/lyase mechanism as proposed by Gacesa <cite> gacesa1987 </cite>. 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 &Delta; 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) <cite> gacesa1987 </cite>. 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 <sup>4</sup>''C''<sub>1</sub> to <sup>1</sup>''C''<sub>4</sub> and changes it from &beta;-<font style="font-feature-settings: 'smcp'">d</font>-mannuronate to &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-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 <cite> ramstad1999, svanem1999, svanem2001, Holtan2006 </cite>. In the lyase mechanism, the second step is a &beta;-elimination of the 4-''O''-glycosidic bond to form a 4-deoxy-<font style="font-feature-settings: 'smcp'">l</font>-''erythro''-hex-4-enepyranosyluronate, called &Delta;, 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 <cite> yoon2001, Ertesvag1998b, Gimmestad2003 </cite>.

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 === Substrate specificities ===
 [[Image:Dermatansulfate.png|thumb|600px|Figure 7. The figure shows a heptamer motif in a dermatan sulfate chain consisting of the possible monomers. GalNAc(4S,6S) represents &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 4,6-''O''-sulfate, GlcA is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronate, GalNAc is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine, IdoA is &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate, GalNAc(4S) is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 4-''O''-sulfate, IdoA(2S) is 2-''O''-sulfo-&alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronate and GalNAc(6S) is &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''N''-acetyl galactosamine 6-''O''-sulfate.]]
-Dermatan sulfate epimerases catalyze the epimerization of &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronic acid (GlcA) to &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronic acid (IdoA) in polymers of glycosaminoglycans <cite>malmstrom1982, maccarana2006, pacheco2009b</cite>, an activity similar to heparin epimerase. The repeating disaccharide unit of dermatan sulfate is 1-3 linked GlcA or IdoA and 1-4 linked &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''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 <cite>trowbridge2002</cite>. 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 <cite>NLM2020</cite>. 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 <cite>trowbridge2002</cite>.
+Dermatan sulfate epimerases catalyze the epimerization of &beta;-<font style="font-feature-settings: 'smcp'">d</font>-glucuronic acid (GlcA) to &alpha;-<font style="font-feature-settings: 'smcp'">l</font>-iduronic acid (IdoA) in polymers of glycosaminoglycans <cite>malmstrom1982, maccarana2006, pacheco2009b</cite>, an activity similar to heparin epimerase. The repeating disaccharide unit of dermatan sulfate is 1,3-linked GlcA or IdoA and 1,4-linked &beta;-<font style="font-feature-settings: 'smcp'">d</font>-''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 <cite>trowbridge2002</cite>. 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 <cite>NLM2020</cite>. 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 <cite>trowbridge2002</cite>.
-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 domain formation. They are found in a variety of animals, including humans <cite>pacheco2009b</cite>.
+Dermatan sulfate consists of block structures of (IdoA-GalNAc)<sub>n</sub>, (GlcA-GalNAc)<sub>n</sub>, 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 <cite>pacheco2009b</cite>.
 Dermatan is a better substrate for the epimerases than chondroitin, and the epimerases are inactive on sulfated substrates <cite>malmstrom1984</cite>. 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 <cite>malmstrom1975, tykesson2018</cite>. The optimal minimal substrate is an octamer <cite>tykesson2016</cite>.

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 === Three-dimensional structures ===
-[[Image:hglce.jpg|thumb|400px|Figure 6. Cartoon representation of the dimeric assembly of human glucuronyl C5-epimerase ([{{PDBlink}}6HZZ PDB ID 6HZZ]) <cite>debarnot2019</cite>. The N-terminal &beta;-hairpin domain is colored in purple, the &beta;-sandwich domain is colored in red and the (&alpha;/&alpha;)<sub>4</sub>-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.]]
+[[Image:hglce.jpg|thumb|600px|Figure 6. Cartoon representation of the dimeric assembly of human glucuronyl C5-epimerase ([{{PDBlink}}6HZZ PDB ID 6HZZ]) <cite>debarnot2019</cite>. The N-terminal &beta;-hairpin domain is colored in purple, the &beta;-sandwich domain is colored in red and the (&alpha;/&alpha;)<sub>4</sub>-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.]]
-In 2015 the first crystal structure was solved, <font style="font-feature-settings: 'smcp'">d</font>-Glucuronyl C5-epimerase from zebrafish ([{{PDBlink}}4PW2 PDB ID 4PW2]) <cite>qin2015</cite>. The protein crystallizes as a homodimer and has three domains: an &alpha;-helical transmembrane region, a &beta;-barrel domain, and a flexible N-terminal loop. Enzyme complexed with an inhibiting heparin hexamer was also crystallized ([{{PDBlink}}4PXQ PDB ID 4PXQ]).
+The first crystal structure was reported in 2015, <font style="font-feature-settings: 'smcp'">d</font>-glucuronyl C5-epimerase from zebrafish ([{{PDBlink}}4PW2 PDB ID 4PW2]) <cite>qin2015</cite>. The protein crystallizes as a homodimer and has three domains: an &alpha;-helical transmembrane region, a &beta;-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 ([{{PDBlink}}4PXQ PDB ID 4PXQ]).
-The next structure to be solved was human <font style="font-feature-settings: 'smcp'">d</font>-Glucuronyl C5-epimerase ([{{PDBlink}}6HZZ PDB ID 6HZZ]) <cite>debarnot2019</cite>. Like the zebrafish enzyme, it is a dimer where each subunit has three domains. The N-terminal domain consists of two antiparallel &beta;-hairpins connected by a helix. This is followed by a &beta;-sandwich domain, and a C-terminal (&alpha;/&alpha;)<sub>4</sub>-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 [{{PDBlink}}6I01 6I01] and [{{PDBlink}}6I02 6I02]) <cite>debarnot2019</cite>.
+The structure of human <font style="font-feature-settings: 'smcp'">d</font>-glucuronyl C5-epimerase has been reported ([{{PDBlink}}6HZZ PDB ID 6HZZ]) <cite>debarnot2019</cite>. Like the zebrafish enzyme, it is a dimer where each subunit has three domains. The N-terminal domain consists of two antiparallel &beta;-hairpins connected by a helix. This is followed by a &beta;-sandwich domain, and a C-terminal (&alpha;/&alpha;)<sub>4</sub>-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 [{{PDBlink}}6I01 6I01] and [{{PDBlink}}6I02 6I02]) <cite>debarnot2019</cite>.
 == Dermatan sulfate C5-epimerase ==

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 === Catalytic reaction and mechanism ===
-The catalytic reaction is thought to follow a similar mechanism to the mannuronan C5-epimerases described [[#Mechanism|above]], and also to that of heparin and dermatan sulfate lyases <cite>Han2009,lunin2004</cite>. A tyrosine is recognized as the proton acceptor and a glutamate as the proton donor. Based on structural analysis of substrate and product complexes it is thought that the reaction proceeds through a neutral enol intermediate, with ring distortion to facilitate epimerization <cite>debarnot2019</cite>. Originally this intermediate was proposed to be an &alpha;-carbanion <cite>prihar1980</cite>, or an enolate as for the other polysaccharide epimerases <cite>qin2015</cite>. Two other tyrosines are shown to be essential for activity, and they are hypothesized to be part of proton relay pathways between the active site and the solvent <cite>qin2015, debarnot2019</cite>. The readdition of the proton is the rate-limiting step <cite>hagner2000b, debarnot2019</cite>. Heparin/heparan epimerases do not require divalent cations for activity, unlike the alginate and dermatan sulfate epimerases <cite>malmstrom1980</cite>. However, one calcium ion is bound to each subunit in the crystal structure of human heparin/heparan epimerase <cite>debarnot2019</cite>. In the crystal structure of zebrafish heparin/heparan epimerase, a water molecule is bound in the same position <cite>qin2015</cite>.
+The catalytic reaction is thought to follow a similar mechanism to the mannuronan C5-epimerases described [[#Mechanism|above]], and also to that of heparin and dermatan sulfate lyases <cite>Han2009,lunin2004</cite>. 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 <cite>debarnot2019</cite>. Originally the intermediate was proposed to be an &alpha;-carbanion/enolate <cite>prihar1980</cite>, as for the other polysaccharide epimerases <cite>qin2015</cite>. 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 <cite>qin2015, debarnot2019</cite>. Protonation of the enol is the rate-limiting step <cite>hagner2000b, debarnot2019</cite>. Heparin/heparan epimerases do not require divalent cations for activity, unlike the alginate and dermatan sulfate epimerases <cite>malmstrom1980</cite>. However, one calcium ion is bound to each subunit in the crystal structure of human heparin/heparan epimerase <cite>debarnot2019</cite>. In the crystal structure of zebrafish heparin/heparan epimerase, a water molecule is bound in the same position <cite>qin2015</cite>.
 === Three-dimensional structures ===

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 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 <font style="font-feature-settings: 'smcp'">l</font>-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 <cite>hagner2004, li2010, sheng2012</cite>.
-An enzyme with a slightly different substrate specificity has been identified from the giant African snail ''Achatina fulica'' <cite>mochizuki2015</cite>. 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.
+An enzyme with a different substrate specificity has been identified from the giant African snail ''Achatina fulica'' <cite>mochizuki2015</cite>. 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 ===