1Dalian Institute of Chemical Physics; Chinese Academy of Sciences; Dalian, PR China

2University of Chinese Academy of Sciences; Beijing, PR China

1Dalian Institute of Chemical Physics; Chinese Academy of Sciences; Dalian, PR China

Alginate lyases catalyze the degradation of alginate, a complex copolymer of α-L-guluronate and its C5 epimer β-D-mannuronate. The enzymes have been isolated from various kinds of organisms with different substrate specificities, including algae, marine mollusks, marine and terrestrial bacteria, and some viruses and fungi. With the progress of structural biology, many kinds of alginate lyases of different polysaccharide lyases families have been characterized by obtaining crystal structures, and the catalytic mechanism has also been elucidated. Combined with various studies, we summarized the source, classification and properties of the alginate lyases from different polysaccharide lyases families. The relationship between substrate specificity and protein sequence was also investigated.

Alginate is the most abundant polysaccharide (about 40% of dry weight) of brown algae, which consists of β-D-mannuronate (M) and α-L-guluronate (G) as monomeric units.1 These units are linked in 3 kinds of different blocks, poly β-D-mannuronate (polyM), poly α-L-guluronate (polyG) and the heteropolymer (polyMG).2 Some bacteria can also synthesize alginates to protect them from detrimental factors such as antibiotics and drought.3,4 Commercial alginates are produced by extraction from biomass of brown algae such as Laminaria hyperborea, Macrocystis pyrifera, Laminaria japonica and etc. Alginate oligosaccharides are depolymerization products of alginate by alginate lyase or physicochemical method. They have attracted increasing attention due to their wide applications in food and pharmaceutical industry.5-7 They can be used as growth promoters for plants and therapeutic agents such as anticoagulants and tumor inhibitors.8-10 They can also induce the cytokine production and regulate the blood sugar as well as lipid.11,12 Due to the high-efficiency and high-specificity, alginate lyases for mild degradation have been the focus for various fields.

Alginate lyase can degrade alginate by β-elimination of glycosidic bonds and produce unsaturated oligosaccharides with double bonds at the non-reducing end.13 A number of alginate lyases have been identified, cloned, purified and characterized from various sources,14-18 including marine and terrestrial bacteria, marine mollusks and algae. They can be classed into 2 groups due to their substrate specificities,13 molecular weights19 and action modes. Now alginate lyases, especially endolytic alginate lyases, have been widely used in the production of alginate oligosaccharides,20 the elucidation of the fine structures of alginate21-23 and the preparation of protoplast of red and brown algae.24-26 Furthermore, alginate lyase also shows great potential application in treatment of cystic fibrosis by degrading the polysaccharide biofilm of bacterium.27-29

Alginate lyases have been isolated from various sources, including marine algae, marine mollusks (Littorina spp., Haliotis spp, Turbo cornutus.), and a wide range of marine and terrestrial bacteria. In addition, some lyases have been isolated from fungi and viruses (Table 1). The alginate lyases can be classified into 2 groups due to their substrate specificities, one is G block-specific lyase (polyG lyase, EC4.2.2.11), and the other is M block-specific lyase (polyM lyase, EC4.2.2.3). This classification has been widely accepted, but some enzymes show activities toward both polyM and polyG,30-33 which may degrade alginate more effectively. In terms of the mode of action, alginate lyase can be grouped into endolytic and exolytic alginate lyase.13 Endolytic alginate lyase cleaves glycosidic bonds inside alginate polymer and releases unsaturated oligosaccharides (di-, tri-, and tetra-saccharides) as main products,30 while exolytic alginate lyase can further degrade oligosaccharides into monomers.31-33 Based on the analysis of hydrophobic cluster of primary structures, alginate lyases can be grouped into 7 families of Polysaccharide Lyase (PL) family (Table 2), PL-5, -6, -7, -14,-15, -17, and -18.34 Most of endolytic bacterial alginate lyases are assigned to PL-5 and PL-7. The most exolytic alginate lyases are grouped into PL-15 and PL-17 families. Most alginate lyases from bacteria are assigned into PL-5, -7, -15, and -17 families. The lyases isolated from marine mollusks and viruses are collected in PL-14 family. The bifunctional alginate lyases belong to PL-18 family, while other lyases are dispersed in other 6 families. According to the 3-dimensional structures, the alginate lyase are grouped into 3 families,35 including parallel β-helix family, (α/α)6 barrel family, jelly-roll family. The alginate lyases of PL-6 belong to parallel β-helix family,36 the PL-5 and -15 members are grouped into (α/α) 6 barrel family,37,38 while -7 and -14 are assigned into jelly-roll family.39,40 The bifunctional alginate lyases of PL-18 are newly characterized and shared a similar sandwich structure.41 Furthermore，the alginate lyases can also be grouped into 3 types based on their molecular masses: small (25–30 kDa), medium-sized (around 40 kDa), and large lyases (>60 kDa).

Alginate lyase of different PL families from different sources and their substrate specificities

Characteristics of alginate lyases from different PL families

The three-dimensional structures of various alginate lyases have been elucidated in Figure 1. The structure of alginate lyase A1-III from Sphingomonas sp. A1 has been resolved complexed with a trisaccharide product.37 The overall structure of A1-III is abundant in helixes and has a deep tunnel-like cleft in a novel (α/α)-barrel structure as the catalytic active domain. The structure presented the possibility that alginate molecules might penetrate into the cleft to interact with the catalytic site of A1-III. This is the only alginate lyase with resolved structure in PL-5. As for PL-7, there are 5 alginate lyases with resolved structures.19,38,42-44 The overall structures of these alginate lyases are all β-sandwich fold like structure with a large active cleft covered by 2 short flexible loops. The overall structure of alginate lyase from Chlorella virus for PL-14 show 2 antiparallel β-sheets with a deep cleft showing a β-jelly roll fold.40 The alginate lyases of PL-17 are known as exolytic alginate lyases and only one has resolved structure with a combination of (α/α) barrel and β-sandwich fold.45 The arrangement of (α/α) helixes produces an open barrel structure and its additional helix serves to maintain rigidity of the barrel. The β-sandwich fold domain is composed of 3 co-planer layers of antiparallel β-strands arranged as sheets and further supported by 4 small helices near the top layer, which interject between the 2 domains. The alginate lyase Aly-SJ02 of PL-18 displays a β-jelly roll scaffold composed mainly of 2 anti-parallels β-sheets.41

The overall structures of alginate lyases from different families.41

Alginate lyase catalyzes the degradation of alginate by β-elimination mechanism, breaking the glycosidic bond between monomers and producing a resulted double bond between the C4 and C5 carbons of the sugar rings. During the elimination, the 4-O-glycosidic bond is eliminated and simultaneously yields oligosaccharides containing 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid as the nonreducing terminal moiety.11 With the 3-dimensional structures of several alginate lyases been resolved,41,45,46 the catalytic mechanism have been elucidated to be 3 steps. The first step is the removal of the negative charge on the carboxyl anion-essentially neutralizing the charge by a salt bridge (Histidine or Lysine) and then a general base-catalyzed abstraction of the proton on C5, where one residue may be required as the proton abstractor and another as the proton donor or the residue act as both proton donor and acceptor. Finally, a transfer of electrons from the carboxyl group forms a double bond between C4 and C5, resulting in the elimination of the 4-O-glycosidic bond.

Alginates are composed of 4 different types of linkage such as M-M, M-G, G-G, and G-M with various extent of each linkage (Fig. 2). Alginate lyases are classified based on their dominant cleaving action on the different types of substrates as shown in Figure 147. The alginate lyases preferring M-rich alginates are assigned into polyM lyases (EC 4. 2. 2. 3), while lyases preferring G-rich alginates are grouped into polyG lyases (EC 4. 2. 2. 11). Although an alginate lyase may be named as polyM lyase or polyG lyase, the enzyme usually displays moderate to low processivity for the other homopolymer. This confusing variable may result from the quality and purity of substrate used for these determinations. These concerns are currently being addressed by using the substrate with high purity and highly sensitive and accurate techniques to analyze the structure of the products. There are several reports of alginate lyases with specific substrate specificities, such as the lyase from ATCC43367 which is reported to cleave only M-M linkages.48 An alginate lyase isolated from marine mollusk Lambis sp displays 100% of activity for polyG while 0% of activity for polyM.49 What is more, an alginate lyase from Klebsiella pneumonia showing activities toward G-blocks and MG-blocks is modified with cleavage specificity for G-G linkage by site-mutagenesis.50 However, some alginate lyases show activities on both polyM and polyG and are regarded as bifunctional lyases, such as alginate lyase AlySJ-02 from Pseudoalteromonas sp. SM0524, AlyPEEC from Pseudoalteromonas sp IAM14594, Aly from Pseudoalteromonas sp. 272, and AlyA from Pseudoalteromonas atlantica AR06.

The substrate specificity of alginate lyase and structures of degradation products. The polysaccharide alginate containing 3 kinds of blocks (M, G, MG) is cleaved to produce a 4-deoxy-L-erythro-hex-4- enepyranosyluronate moiety (open triangle) at the newly formed non-reducing end of the product.47

The alginate lyases of PL7 family have been well investigated. There are 25 characterized alginate lyases in PL-7 family, and 5 of them have been investigated for structure-function relationship by obtaining crystal structures. The structural basis for depolymerization of alginate lyase has been elucidated.51 As shown in Figure 3, the alginate lyases contain 3 highly conserved regions, (R/E) (S/T/N) EL, Q (I/V) H, YFKAG (V/I) YNQ. According to the crystal structures and the sequence analysis of several PL family 7 lyases, those conserved regions form the cavity composed of a jelly-roll β-sandwich structure, and the cavity is assumed to bind a suitable substrate. Thus, conserved amino acids residues are thought to play an important role in catalytic activity or folding of the structure. It has been reported that Y195, H119, Q117 and R72 may be involved in the catalytic site for ALY-1.52 Therefore, the amino acids in catalytic and substrate binding sites would be highly conserved depending on the substrate specificity. The polyM-specific alginate lyases contain QVH in conserved regions and polyG-specific enzymes involve QIH in conserved regions, while the polyMG lyases include QIH in the conserved regions. In addition, the substitution of hydrophobic residues in the isoleucine site of domain QIH could have enormous influence on the high affinity to polyG block. For this perspective, the substrate specificity is determined by the isoleucine of QIH domain and valine of QVH domain. The isoleucine of QIH domain is confirmed to be indispensible for reorganization of the polyG or G-G bond.53 So, if an alginate lyase with unknown substrate specificity contained isoleucine or valine in Q (I/V) H region, this enzyme could degrade polyM or polyG and polyMG, respectively. The region YFKAG (V/I) YNQ has been reported from various alginate lyases with different specificities, indicating that this region may be of functional or structural importance for alginate degradation independent of substrate preference. Considering that the alginate lyase totally lost activity with deletion of this region, the results suggest that the region may be needed to catalyze alginate degradation.

Multiple alignment of amino acid sequences of alginate lyases of PL 7 family. The conserved residues were overlaid with red and 3 highly conserved regions-(R/E) (S/T/N) EL, Q (I/V) H, YFKAG (V/I) YNQ were boxed in blue.

Depolymerized alginate produced by enzymatic hydrolysis with low DP possesses various kinds of biological activities.8-14 Oligomeric alginates obtained from lyase degradation of alginate act as oligosaccharines and regulate physiological processes in plants and microorganisms, such as promoting the growth of Bifidobacterium spp., enhancing the germination and shoot elongation in plants. The oligosaccharides exhibit many physiological activities, such as antitumor, stimulating production of cytokines, regulating the blood lipids and sugars. The efficient alginate lyases w are key tools for production of functional oligosaccharides from alginate.

The alginate lyases with different substrate specificities are invaluable tools for determining sequence of substrate and preparing the oligosaccharides with specific structures. Alginate lyases possess different substrate specificities depending on the differences in amino acids and distributing of monosaccharide residues in substrate. Various enzymes could recognize 4 different types of linkages such as M-M, M-G, G-M, and G-G. The alginate lyases of PL-5 family prefer to degrade polyM substrate. The AlgA from Pseudomonas sp E03 hydrolyzes the polyM substrate in an endolytic manner and releases a range of oligomannuronate with low DP.11 The AlxM from Photobacterium sp. ATCC43367 is also a polyM-specific alginate lyase.48 And a polyM lyase from Sargassum fluitans specifically depolymerize polyM.54 The alginate lyases specific for G-G linkage have not been described except for a mutant enzyme with cleavage specificity for G-G linkage.14 This G-G linkage specific enzyme is constructed by mutating the gene encoding Klebsiella pneumonia AlyA, which cleaves both G-G and G-M linkages. The enzyme isolated from Klebsiella pneumonia shows activity only toward G-G linkage.50 The polyMG-specific alginate lyase from Stenotrophomas maltophilia KJ-2 preferably degrades the glycosidic bond in M-G linkage than that in G-M linkage.55 The alginate lyases have been used to analyze alginate fine structure to understand how chemical composition influences the physical properties of alginate. The combination of alginate lyases with different substrate specificities has used for determining the sequence and block distribution of alginate.56

In addition, the enzymes with strict substrate specificities could be used for preparation alginate with specific structures. The AlxMB can be used for preparation of homopolymannuronate blocks and strictly alternating sequences of mannuronic and guluronic acids due to its strict substrate specificity toward polyM blocks.57 A simple method for preparation of polyG blocks has been developed by using a polyG lyase from Flavobacterium multivolum.58 The enzyme could degrade both G- and MG- blocks but M blocks. The guluronan lyase from Klebsiella aeruginosa has no action on polyM, but can readily cleave G-G linkages in guluronan. Based on the substrate specificity of this enzyme, it has been used for determining the activity of C-5 epimerase.59

Recently, the alginate lyases have been used to treat the cystic fibrosis in combination with antibiotics.27-29 When alginate lyase is co-administered with antibiotics such as gentamycin, the killing efficiency of mucoid Pseudomonas aeruginosa in respiratory tract increases. Therefore, the enzymes are expected to become useful agents for the treatment of bacterial mucoid biofilm-dependent diseases. What is more, the alginate lyases are also used to prepare the protoplast of algae for gene engineering and study Fucus cell wall development. With the consumption of fossil source, the alginate, being regarded as a renewable source for the production of bioethanol, has obtained increased attention. The saccharification of alginate for production of bioethanol requires synergistic effects of alginate lyases with endo- and exo- action modes. The engineered microbial platforms for direct production of bioethanol from alginate have been reported.60-62

In conclusion, the enzymes are important tools in a broad spectrum of biological roles and applications. Alginate lyases with different substrate specificities can be used to produce oligosaccharides with various biological functionalities. Moreover, they can also be used for determination of the fine structure of alginate and the preparation of tailor-made alginate. Furthermore, alginate lyases with activities toward acetylated alginate can be utilized for the treatment of cystic fibrosis in combination with antibiotics. Therefore, due to the various activities and biological functionalities, alginate lyases have been widely applied. And with the continuing developments for screening and characterizing the enzymes, the applications of alginate lyases will become increasing wider in the near future.

The alginate lyases have important roles and biotechnological applications. Especially, the enzymes were key tools for production of oligosaccharides from alginate and determination of the fine structure of alginate. In addition, the alginate lyases play essential role in saccharification of the acidic polysaccharides for production of bioethanol with synergistic effect of endo- and exo-type alginate lyases. There were several hundred kinds of alginate lyases from various sources have been isolated and characterized. These enzymes differed from each other in substrate specificities, properties (including activities and structures) and degradation products. With the progress of structural biology, many kinds of alginate lyases of different PL families have been characterized by obtaining crystal structures, and the catalytic mechanism has also been elucidated. In future, screening and characterization of novel enzymes with high activities and broad substrate specificities would enhance and expand the utilization of the lyases to produce oligosaccharides with novel structures and biological activities for applications in various fields.

No potential conflicts of interest were disclosed.

The research in our lab mentioned in this paper was supported by the National Key Technology Support Program (2013BAB01B01), Special Fund for Marine Scientific Research (201305015-2), National Natural Science Foundation of China (31370811) and Development Fund for Collaborative Innovation Center of Glycoscience of Shandong University.