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.

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 [81]. 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.

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 [82, 83]. The motifs stabilize the fold by binding calcium ions tightly [84], 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, 85].

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].

The parallel β-helix fold found in the active modules of the epimerases was first encountered in another enzyme active on polyanionic substrate, namely the pectate lyase C of Erwinia chrysanthemi [86]. It is also called a β-solenoid-type fold as it consists of repeated β strands, and it is a type L β-solenoid [87]. In the epimerases it 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 (GH28, GH49, GH55, GH82 and GH87 in the CAZy database [88]) and of polysaccharide lyases (PL1, PL3, PL6 and PL9 [89]): alginate lyases [77], dextranases [90], rhamnogalacturonases [91], polygalacturonase [92], β-1,3-glucanases [93], ι-carrageenases [94], endorhamnosidases [95], pectate and pectin lyases [86, 96, 97, 98] and chondroitin B (dermatan sulfate) lyases [75]. Since these enzymes have a high degree of structural similarity, it is thought that at least some of them diverged from a common ancestor [99], although convergent evolution is not unlikely [87].

References

1. Haug A and Larsen B. (1969). Biosynthesis of alginate. Epimerisation of D-mannuronic to L-guluronic acid residues in the polymer chain. Biochim Biophys Acta. 1969;192(3):557-9. DOI:10.1016/0304-4165(69)90414-0 |
2. Larsen B and Haug A. (1971). Biosynthesis of alginate. 1. Composition and structure of alginate produced by Azotobacter vinelandii (Lipman). Carbohydr Res. 1971;17(2):287-96. DOI:10.1016/s0008-6215(00)82536-7 |
3. Haug A and Larsen B. (1971). Biosynthesis of alginate. II. Polymannuronic acid C-5-epimerase from Azotobacter vinelandii (Lipman). Carbohydr Res. 1971;17(2):297-308. DOI:10.1016/s0008-6215(00)82537-9 |

4. Stanford, Edw C C. (1883) On algin: a new substance obtained from some of the commoner species of marine algae. R. Anderson. NLM ID: 101217546

5. Gorin, P. A. J. and Spencer, J. F. T. (1966) Exocellular alginic acid from Azotobacter vinelandii. Canadian Journal of Chemistry vol. 44, no. 9., pp. 993-998. DOI: 10.1139/v66-147

6. Linker A and Jones RS. (1966). A new polysaccharide resembling alginic acid isolated from pseudomonads. J Biol Chem. 1966;241(16):3845-51. | Google Books | Open Library
7. Govan JR, Fyfe JA, and Jarman TR. (1981). Isolation of alginate-producing mutants of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas mendocina. J Gen Microbiol. 1981;125(1):217-20. DOI:10.1099/00221287-125-1-217 |

8. Okazaki, M., K. and Furuya, K. Tsukayam and K. Nisizawa. (1982) Isolation and Identification of Alginic Acid from a Calcareous Red Alga Serraticardia maxima. Botanica Marina, vol. 25, no. 3., pp. 123-131. [1]

9. Painter, Terence J. (1983) Chapter 4 - Algal Polysaccharides. Edited by Gerald O. Aspinall. The Polysaccharides. New York: Academic Press. DOI: 10.1016/B978-0-12-065602-8.50009-1

10. Campos M, Martínez-Salazar JM, Lloret L, Moreno S, Núñez C, Espín G, and Soberón-Chávez G. (1996). Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii. J Bacteriol. 1996;178(7):1793-9. DOI:10.1128/jb.178.7.1793-1799.1996 |
11. Pier GB, Coleman F, Grout M, Franklin M, and Ohman DE. (2001). Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect Immun. 2001;69(3):1895-901. DOI:10.1128/IAI.69.3.1895-1901.2001 |
12. Harmsen M, Yang L, Pamp SJ, and Tolker-Nielsen T. (2010). An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal. FEMS Immunol Med Microbiol. 2010;59(3):253-68. DOI:10.1111/j.1574-695X.2010.00690.x |

13. Hirst, E. L. and Jones, J. K. N and Jones, Winifred Osman. (1939) 389. The structure of alginic acid. Part I [in en]. Journal of the Chemical Society, The Royal Society of Chemistry. Vol. 0, pp. 1880–1885. DOI: 10.1039/JR9390001880

14. FISCHER FG and DORFEL H. (1955). [Polyuronic acids in brown algae]. Hoppe Seylers Z Physiol Chem. 1955;302(4-6):186-203. | Google Books | Open Library

15. Drummond, D W and Hirst, E L and Percival, Elizabeth. (1962) 232. The constitution of alginic acid. Journal of the Chemical Society, The Royal Society of Chemistry. Vol. 0, pp. 1208–1216. DOI: 10.1039/JR9620001208

16. Ertesvåg H, Høidal HK, Schjerven H, Svanem BI, and Valla S. (1999). Mannuronan C-5-epimerases and their application for in vitro and in vivo design of new alginates useful in biotechnology. Metab Eng. 1999;1(3):262-9. DOI:10.1006/mben.1999.0130 |
17. Lin TY and Hassid WZ. (1966). Pathway of algnic acid synthesis in the marine brown alga, Fucus gardneri Silva. J Biol Chem. 1966;241(22):5284-97. | Google Books | Open Library
18. Franklin MJ, Chitnis CE, Gacesa P, Sonesson A, White DC, and Ohman DE. (1994). Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase. J Bacteriol. 1994;176(7):1821-30. DOI:10.1128/jb.176.7.1821-1830.1994 |

19. Haug, Arne and Larsen, Bjørn and Smidsrød, Olav. (1966) A study on the constitution of alginic acidby partial acid hydrolysis. Acta Chemica Scandinavica, vol. 5 (July), pp. 271–277. [1]

20. Haug, Arne and Larsen, Bjørn and Smidsrød, Olav. (1967) Studies on the Sequence of Uronic Acid Residues in Alginic Acid. Acta Chemica Scandinavica, vol. 21, pp. 691–794. [1]

21. Skjåk-Bræk, Gudmund and Larsen, Bjørn and Grasdalen, Hans. (1985) The role of O-acetyl groupsin the biosynthesis of alginate by Azotobacter vinelandii. Carbohydrate Research, vol. 145, no. 1, pp. 169–174. [1]

22. Kristiansen, Kåre A and Schirmer, Bjørn C and Aachmann, Finn L. and Skjåk-Bræk, Gudmund and Draget, Kurt I. and Christensen, Bjørn E. (2009) Novel alginates prepared by independent control of chain stiff-ness and distribution of G-residues: Structure and gelling properties. Carbohydrate Polymers, vol. 77, no.4, pp. 725–735. [1]

23. Madgwick J, Haug A, and Larsen B. (1973). Polymannuronic acid 5-epimerase from the marine alga Pelvetia canaliculata (L.) Dcne. et Thur. Acta Chem Scand. 1973;27(9):3592-4. DOI:10.3891/acta.chem.scand.27-3592 |
24. Fischl R, Bertelsen K, Gaillard F, Coelho S, Michel G, Klinger M, Boyen C, Czjzek M, and Hervé C. (2016). The cell-wall active mannuronan C5-epimerases in the model brown alga Ectocarpus: From gene context to recombinant protein. Glycobiology. 2016;26(9):973-983. DOI:10.1093/glycob/cww040 |
25. Nyvall P, Corre E, Boisset C, Barbeyron T, Rousvoal S, Scornet D, Kloareg B, and Boyen C. (2003). Characterization of mannuronan C-5-epimerase genes from the brown alga Laminaria digitata. Plant Physiol. 2003;133(2):726-35. DOI:10.1104/pp.103.025981 |
26. Chitnis CE and Ohman DE. (1990). Cloning of Pseudomonas aeruginosa algG, which controls alginate structure. J Bacteriol. 1990;172(6):2894-900. DOI:10.1128/jb.172.6.2894-2900.1990 |
27. Morea A, Mathee K, Franklin MJ, Giacomini A, O'Regan M, and Ohman DE. (2001). Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens. Gene. 2001;278(1-2):107-14. DOI:10.1016/s0378-1119(01)00685-0 |
28. Ertesvåg H, Doseth B, Larsen B, Skjåk-Braek G, and Valla S. (1994). Cloning and expression of an Azotobacter vinelandii mannuronan C-5-epimerase gene. J Bacteriol. 1994;176(10):2846-53. DOI:10.1128/jb.176.10.2846-2853.1994 |
29. Ertesvåg H, Høidal HK, Hals IK, Rian A, Doseth B, and Valla S. (1995). A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii. Mol Microbiol. 1995;16(4):719-31. DOI:10.1111/j.1365-2958.1995.tb02433.x |
30. Rehm BH, Ertesvåg H, and Valla S. (1996). A new Azotobacter vinelandii mannuronan C-5-epimerase gene (algG) is part of an alg gene cluster physically organized in a manner similar to that in Pseudomonas aeruginosa. J Bacteriol. 1996;178(20):5884-9. DOI:10.1128/jb.178.20.5884-5889.1996 |
31. Svanem BI, Skjåk-Braek G, Ertesvåg H, and Valla S. (1999). Cloning and expression of three new Aazotobacter vinelandii genes closely related to a previously described gene family encoding mannuronan C-5-epimerases. J Bacteriol. 1999;181(1):68-77. DOI:10.1128/JB.181.1.68-77.1999 |
32. Gimmestad M, Sletta H, Ertesvåg H, Bakkevig K, Jain S, Suh SJ, Skjåk-Braek G, Ellingsen TE, Ohman DE, and Valla S. (2003). The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation. J Bacteriol. 2003;185(12):3515-23. DOI:10.1128/JB.185.12.3515-3523.2003 |
33. Ertesvåg H and Valla S. (1999). The A modules of the Azotobacter vinelandii mannuronan-C-5-epimerase AlgE1 are sufficient for both epimerization and binding of Ca2+. J Bacteriol. 1999;181(10):3033-8. DOI:10.1128/JB.181.10.3033-3038.1999 |

34. Ramstad, Marit Valeur and Ellingsen, Trond E. and Josefsen, Kjell D. and Høidal, Hilde K. and Valla, Svein and Skjåk-Bræk, Gudmund and Levine, David W. (1999) Properties and action pattern of the recombinant mannuronan C-5-epimerase AlgE2. Enzyme and Microbial Technology, vol. 24, no. 10, pp. 636–646. [1]

35. Holtan S, Bruheim P, and Skjåk-Braek G. (2006). Mode of action and subsite studies of the guluronan block-forming mannuronan C-5 epimerases AlgE1 and AlgE6. Biochem J. 2006;395(2):319-29. DOI:10.1042/BJ20051804 |
36. Buchinger E, Knudsen DH, Behrens MA, Pedersen JS, Aarstad OA, Tøndervik A, Valla S, Skjåk-Bræk G, Wimmer R, and Aachmann FL. (2014). Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii. J Biol Chem. 2014;289(45):31382-96. DOI:10.1074/jbc.M114.567008 |
36. Buchinger E, Knudsen DH, Behrens MA, Pedersen JS, Aarstad OA, Tøndervik A, Valla S, Skjåk-Bræk G, Wimmer R, and Aachmann FL. (2014). Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii. J Biol Chem. 2014;289(45):31382-96. DOI:10.1074/jbc.M114.567008 |
37. Campa C, Holtan S, Nilsen N, Bjerkan TM, Stokke BT, and Skjåk-Braek G. (2004). Biochemical analysis of the processive mechanism for epimerization of alginate by mannuronan C-5 epimerase AlgE4. Biochem J. 2004;381(Pt 1):155-64. DOI:10.1042/BJ20031265 |
38. Høidal HK, Ertesvåg H, Skjåk-Braek G, Stokke BT, and Valla S. (1999). The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism. J Biol Chem. 1999;274(18):12316-22. DOI:10.1074/jbc.274.18.12316 |
39. Hartmann M, Holm OB, Johansen GA, Skjåk-Braek G, and Stokke BT. (2002). Mode of action of recombinant Azotobacter vinelandii mannuronan C-5 epimerases AlgE2 and AlgE4. Biopolymers. 2002;63(2):77-88. DOI:10.1002/bip.10017 |

40. Sletmoen, Marit and Skjåk-Bræk, Gudmund and Stokke, Bjørn T. (2004) Single-molecular Pair Unbinding Studies of Mannuronan C-5 Epimerase AlgE4 and Its Polymer Substrate, Biomacromolecules, American Chemical Society, vol. 5, no.4, pp. 1288–1295. DOI: 10.1021/BM0345211

41. Håti AG, Aachmann FL, Stokke BT, Skjåk-Bræk G, and Sletmoen M. (2015). Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy. PLoS One. 2015;10(10):e0141237. DOI:10.1371/journal.pone.0141237 |
43. Svanem BI, Strand WI, Ertesvag H, Skjåk-Braek G, Hartmann M, Barbeyron T, and Valla S. (2001). The catalytic activities of the bifunctional Azotobacter vinelandii mannuronan C-5-epimerase and alginate lyase AlgE7 probably originate from the same active site in the enzyme. J Biol Chem. 2001;276(34):31542-50. DOI:10.1074/jbc.M102562200 |

44. Yoon, Hye-Jin and Hashimoto, Wataru and Miyake, Osamu and Murata, Kousaku and Mikami, Bunzo. (2001) Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0 Å resolution, Journal of Molecular Biology, vol. 307, no. 1 pp. 9–16. DOI: 10.1006/jmbi.2000.4509

45. Ertesvåg H, Erlien F, Skjåk-Braek G, Rehm BH, and Valla S. (1998). Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase. J Bacteriol. 1998;180(15):3779-84. DOI:10.1128/JB.180.15.3779-3784.1998 |
46. DISCHE Z (1947). A new specific color reaction of hexuronic acids. J Biol Chem. 1947;167(1):189-98. | Google Books | Open Library
47. Knutson CA and Jeanes A. (1968). A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem. 1968;24(3):470-81. DOI:10.1016/0003-2697(68)90154-1 |
48. Knutson CA and Jeanes A. (1968). Determination of the composition of uronic acid mixtures. Anal Biochem. 1968;24(3):482-90. DOI:10.1016/0003-2697(68)90155-3 |

49. Skjåk-Bræk, Gudmund, and Larsen, Bjørn. (1982) Biosynthesis of Alginate Part 5. A New Assay for Mannuronan C-5-Epimerase Activity, Carbohydrate Research, vol. 103, no.1, pp. 133–136. DOI: 10.1016/S0008-6215(82)80013-X

50. Skjåk-Bræk, Gudmund and Larsen, Bjørn. (1985) Biosynthesis of alginate: Purification and characterisation of mannuronan C-5-epimerase from Azotobacter vinelandii, Carbohydrate Research, vol. 139 (June), pp. 273–283. DOI:10.1016/0008-6215(85)90026-6

51. Currie, Andrew J., and Turvey, James R. (1982) An enzymic method for the assay of d-mannuronanC-5 epimerase activity. Carbohydrate Research, vol. 107, no. 1, pp. 156–159. DOI: 10.1016/S0008-6215(00)80786-7

52. PREISS J and ASHWELL G. (1962). Alginic acid metabolism in bacteria. I. Enzymatic formation of unsaturated oligosac-charides and 4-deoxy-L-erythro-5-hexoseulose uronic acid. J Biol Chem. 1962;237:309-16. | Google Books | Open Library
53. Boyd J and Turvey JR. (1977). Isolation of poly-alpha-L-guluronate lyase from Klebsiella aerogenes. Carbohydr Res. 1977;57:163-71. DOI:10.1016/s0008-6215(00)81928-x |

54. Grasdalen, Hans and Larsen, Bjørn and Smidsrød, Olav. (1977) ¹³C-N.m.r. studies of alginate. Carbohydrate Research, vol. 56, no. 2, pp. C11–C15. DOI: 10.1016/S0008-6215(00)83369-8

55. Grasdalen, Hans and Larsen, Bjørn and Smidsrød, Olav. (1981) ¹³C-n.m.r. studies of monomeric composition and sequence in alginate. Carbohydrate Research, vol. 89, no. 2, pp. 179–191. DOI: 10.1016/S0008-6215(00)85243-X

56. Grasdalen, Hans and Larsen, Bjørn and Smidsrød, Olav. (1979) A p.m.r. study of the composition and sequence of uronate residues in alginates. Carbohydrate Research, vol. 68, no. 1, pp. 23–31. DOI: 10.1016/S0008-6215(00)84051-3

57. Grasdalen, Hans. (1983) High-field, ¹H-n.m.r. spectroscopy of alginate: sequential structure andlinkage conformations. Carbohydrate Research, vol. 118 (July), pp. 255–260. DOI: 10.1016/0008-6215(83)88053-7

58. Hartmann M, Duun AS, Markussen S, Grasdalen H, Valla S, and Skjåk-Braek G. (2002). Time-resolved 1H and 13C NMR spectroscopy for detailed analyses of the Azotobacter vinelandii mannuronan C-5 epimerase reaction. Biochim Biophys Acta. 2002;1570(2):104-12. DOI:10.1016/s0304-4165(02)00195-2 |
59. Tøndervik A, Klinkenberg G, Aachmann FL, Svanem BI, Ertesvåg H, Ellingsen TE, Valla S, Skjåk-Bræk G, and Sletta H. (2013). Mannuronan C-5 epimerases suited for tailoring of specific alginate structures obtained by high-throughput screening of an epimerase mutant library. Biomacromolecules. 2013;14(8):2657-66. DOI:10.1021/bm4005194 |
60. Stanisci A, Tøndervik A, Gaardløs M, Lervik A, Skjåk-Bræk G, Sletta H, and Aachmann FL. (2020). Identification of a Pivotal Residue for Determining the Block Structure-Forming Properties of Alginate C-5 Epimerases. ACS Omega. 2020;5(8):4352-4361. DOI:10.1021/acsomega.9b04490 |

61. Morris, Edwin R., Rees, David A. and Thom, David. (1973) Characterization of polysaccharide structure and interactions by circular dichroism: order–disorder transition in the calcium alginate system. Journal of the Chemical Society, The Royal Society of Chemistry, Chemical Communications, no. 7, pp. 245–246. DOI: 10.1039/C39730000245

62. Donati, Ivan and Gamini, Amelia and Skjåk-Bræk, Gudmund and Vetere, Amedeo and Campa, Cristiana and Coslovi, Anna and Paoletti, Sergio. (2003) Determination of the diadic composition of alginate by means of circular dichroism: a fast and accurate improved method. Carbohydrate Research, vol. 338, no. 10, pp. 1139–1142. DOI: 10.1016/S0008-6215(03)00094-6

63. Reese, Johanna de and Sperl, Nadine and Schmid, Jochen and Sieber, Volker and Plank, Johann. (2015) Effect of biotechnologically modified alginates on LDH structures. Bioinspired, Biomimetic and Nanobiomaterials, vol. 4, no. 3, pp. 174–186. DOI: 10.1680/jbibn.14.00032

64. Tøndervik A, Klinkenberg G, Aarstad OA, Drabløs F, Ertesvåg H, Ellingsen TE, Skjåk-Bræk G, Valla S, and Sletta H. (2010). Isolation of mutant alginate lyases with cleavage specificity for di-guluronic acid linkages. J Biol Chem. 2010;285(46):35284-92. DOI:10.1074/jbc.M110.162800 |
65. Campa C, Oust A, Skjåk-Braek G, Paulsen BS, Paoletti S, Christensen BE, and Ballance S. (2004). Determination of average degree of polymerisation and distribution of oligosaccharides in a partially acid-hydrolysed homopolysaccharide: a comparison of four experimental methods applied to mannuronan. J Chromatogr A. 2004;1026(1-2):271-81. DOI:10.1016/j.chroma.2003.11.045 |

66. Ballance, Simon and Holtan, Synnøve and Aarstad, Olav Andreas and Sikorski, Pawel and Skjåk-Bræk, Gudmund and Christensen, Bjørn E. (2005) Application of high-performance anion-exchange chromatography with pulsed amperometric detection and statistical analysis to study oligosaccharide distributions – a complementary method to investigate the structure and some properties of alginates. Journal of Chromatography A, vol. 1093, no. 1, pp. 59–68. DOI: 10.1016/j.chroma.2005.07.051

67. Aarstad OA, Tøndervik A, Sletta H, and Skjåk-Bræk G. (2012). Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes. Biomacromolecules. 2012;13(1):106-16. DOI:10.1021/bm2013026 |
68. Douthit SA, Dlakic M, Ohman DE, and Franklin MJ. (2005). Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed beta-helix. J Bacteriol. 2005;187(13):4573-83. DOI:10.1128/JB.187.13.4573-4583.2005 |
69. Rozeboom HJ, Bjerkan TM, Kalk KH, Ertesvåg H, Holtan S, Aachmann FL, Valla S, and Dijkstra BW. (2008). Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii. J Biol Chem. 2008;283(35):23819-28. DOI:10.1074/jbc.M804119200 |
70. Wolfram F, Kitova EN, Robinson H, Walvoort MT, Codée JD, Klassen JS, and Howell PL. (2014). Catalytic mechanism and mode of action of the periplasmic alginate epimerase AlgG. J Biol Chem. 2014;289(9):6006-19. DOI:10.1074/jbc.M113.533158 |

71. Jurrus, Elizabeth and Engel, Dave and Star, Keith and Monson, Kyle and Brandi, Juan and Felberg, Lisa E. and Brookes, David H. et al. (2018) Improvements to the APBS biomolecular solvation software suite. Protein Science, vol. 27, no. 1, pp. 112–128. DOI: 10.1002/pro.3280

72. Schrödinger, LLC. (2018) The PyMOL Molecular Graphics System, Version 2.2.3.

73. Ofstad, Ragni and Larsen, Bjørn. (1981) The effect of calcium ion concentration on poly-D-mannuronate C-5 epimerase. Proceedings of the 10th International Seaweed Symposium, edited by Tore Levring, pp. 485–493.

74. Ertesvåg H, Høidal HK, Skjåk-Braek G, and Valla S. (1998). The Azotobacter vinelandii mannuronan C-5-epimerase AlgE1 consists of two separate catalytic domains. J Biol Chem. 1998;273(47):30927-32. DOI:10.1074/jbc.273.47.30927 |
75. Huang W, Matte A, Li Y, Kim YS, Linhardt RJ, Su H, and Cygler M. (1999). Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution. J Mol Biol. 1999;294(5):1257-69. DOI:10.1006/jmbi.1999.3292 |
76. Michel G, Pojasek K, Li Y, Sulea T, Linhardt RJ, Raman R, Prabhakar V, Sasisekharan R, and Cygler M. (2004). The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery. J Biol Chem. 2004;279(31):32882-96. DOI:10.1074/jbc.M403421200 |
77. Xu F, Dong F, Wang P, Cao HY, Li CY, Li PY, Pang XH, Zhang YZ, and Chen XL. (2017). Novel Molecular Insights into the Catalytic Mechanism of Marine Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6. J Biol Chem. 2017;292(11):4457-4468. DOI:10.1074/jbc.M116.766030 |
78. Stender EGP, Dybdahl Andersen C, Fredslund F, Holck J, Solberg A, Teze D, Peters GHJ, Christensen BE, Aachmann FL, Welner DH, and Svensson B. (2019). Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus. J Biol Chem. 2019;294(47):17915-17930. DOI:10.1074/jbc.RA119.010206 |
79. Aachmann FL, Svanem BI, Güntert P, Petersen SB, Valla S, and Wimmer R. (2006). NMR structure of the R-module: a parallel beta-roll subunit from an Azotobacter vinelandii mannuronan C-5 epimerase. J Biol Chem. 2006;281(11):7350-6. DOI:10.1074/jbc.M510069200 |

80. Sletmoen, Marit and Skjåk-Bræk, Gudmund and Stokke, Bjørn T. (2005) Mapping enzymatic functionalities of mannuronan C-5 epimerases and their modular units by dynamic force spectroscopy. Carbohydrate Research, vol. 340, no.18, pp. 2782–2795. DOI: 10.1016/J.CARRES.2005.09.020

81. Yoder MD, Lietzke SE, and Jurnak F. (1993). Unusual structural features in the parallel beta-helix in pectate lyases. Structure. 1993;1(4):241-51. DOI:10.1016/0969-2126(93)90013-7 |
82. Welch RA (1991). Pore-forming cytolysins of gram-negative bacteria. Mol Microbiol. 1991;5(3):521-8. DOI:10.1111/j.1365-2958.1991.tb00723.x |

83. Lilie, Hauke and Haehnel, Wolfgang and Rudolph, Rainer and Baumann, Ulrich. (2000) Folding of a synthetic parallel β-roll protein. FEBS Letters, vol. 470, no. 2, pp. 173–177. DOI: 10.1016/S0014-5793(00)01308-9

84. Jenkins, John and Pickersgill, Richard. (2001) The architecture of parallel β-helices and related folds. Progress in Biophysics and Molecular Biology, vol. 77, no. 2, pp. 111–175. DOI: 10.1016/S0079-6107(01)00013-X

85. Gimmestad M, Steigedal M, Ertesvåg H, Moreno S, Christensen BE, Espín G, and Valla S. (2006). Identification and characterization of an Azotobacter vinelandii type I secretion system responsible for export of the AlgE-type mannuronan C-5-epimerases. J Bacteriol. 2006;188(15):5551-60. DOI:10.1128/JB.00236-06 |
86. Yoder MD, Keen NT, and Jurnak F. (1993). New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science. 1993;260(5113):1503-7. DOI:10.1126/science.8502994 |
88. Rigden DJ and Franco OL. (2002). Beta-helical catalytic domains in glycoside hydrolase families 49, 55 and 87: domain architecture, modelling and assignment of catalytic residues. FEBS Lett. 2002;530(1-3):225-32. DOI:10.1016/s0014-5793(02)03490-7 |

89. Garron, M.-L. and Cygler, M. (2010) Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology, vol. 20, no. 12, pp. 1547–1573. DOI: 10.1093/glycob/cwq122

90. Larsson, Anna M and Andersson, Rolf and Ståhlberg, Jerry and Kenne, Lennart and Jones, T. Alwyn. (2003) Dextranase from Penicillium minioluteum: Reaction Course, Crystal Structure, and Product Complex. Structure, vol. 11, no. 9, pp. 1111–1121. DOI: 10.1016/S0969-2126(03)00147-3

91. Petersen TN, Kauppinen S, and Larsen S. (1997). The crystal structure of rhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel beta helix. Structure. 1997;5(4):533-44. DOI:10.1016/s0969-2126(97)00209-8 |
92. Pickersgill R, Smith D, Worboys K, and Jenkins J. (1998). Crystal structure of polygalacturonase from Erwinia carotovora ssp. carotovora. J Biol Chem. 1998;273(38):24660-4. DOI:10.1074/jbc.273.38.24660 |
93. Ishida T, Fushinobu S, Kawai R, Kitaoka M, Igarashi K, and Samejima M. (2009). Crystal structure of glycoside hydrolase family 55 {beta}-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J Biol Chem. 2009;284(15):10100-9. DOI:10.1074/jbc.M808122200 |
94. Michel G, Chantalat L, Fanchon E, Henrissat B, Kloareg B, and Dideberg O. (2001). The iota-carrageenase of Alteromonas fortis. A beta-helix fold-containing enzyme for the degradation of a highly polyanionic polysaccharide. J Biol Chem. 2001;276(43):40202-9. DOI:10.1074/jbc.M100670200 |
95. Steinbacher S, Seckler R, Miller S, Steipe B, Huber R, and Reinemer P. (1994). Crystal structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer. Science. 1994;265(5170):383-6. DOI:10.1126/science.8023158 |

96. Mayans, O. and Scott, M. and Connerton, I. and Gravesen, T. and Benen, J. and Visser, J. and Pickersgill, R. and Jenkins, J. (1997) Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases. Structure, vol. 5, no. 5, pp. 677–689. DOI: 10.1016/S0969-2126(97)00222-0

97. Akita M, Suzuki A, Kobayashi T, Ito S, and Yamane T. (2001). The first structure of pectate lyase belonging to polysaccharide lyase family 3. Acta Crystallogr D Biol Crystallogr. 2001;57(Pt 12):1786-92. DOI:10.1107/s0907444901014482 |
98. Jenkins J, Shevchik VE, Hugouvieux-Cotte-Pattat N, and Pickersgill RW. (2004). The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi. J Biol Chem. 2004;279(10):9139-45. DOI:10.1074/jbc.M311390200 |

99. Jenkins, John and Mayans, Olga and Pickersgill, Richard. (1998) Structure and Evolution of Parallel β-Helix Proteins. Journal of Structural Biology, vol. 122, nos. 1-2, pp. 236–246. DOI: 10.1006/JSBI.1998.3985