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== Mechanism ==
 
== Mechanism ==
[[Image:AlyPL7 MultipleSequenceAlignment.JPG|thumb|400px|'''Figure 1. Multiple protein alignment of Aly PL7 ''' as well as a secondary structure prediction of the crystallized PL7 from ''Klabsiella pneumoniae'' ([{{PDBlink}}4OZX PDB ID 4OZX]). Conserved residues in the homologues are colored in red and (putative) catalytic residues are indicated by a star. MSA was done using Espript3.0 <cite>Robert2014</cite>.]]
+
[[Image:AlyPL7 MultipleSequenceAlignment.JPG|thumb|400px|'''Figure 1. Multiple protein sequence alignment of Aly PL7 ''' as well as a secondary structure prediction with the crystallized PL7 from ''Klabsiella pneumoniae'' ([{{PDBlink}}4OZX PDB ID 4OZX]). Conserved residues in the homologues are colored in red and (putative) catalytic residues are indicated by a star. The multiple protein sequence alignment was done with Espript3.0 <cite>Robert2014</cite>.]]
Alginate lyases (Alys) of all families, PL5-7, PL14-15, PL17-18, catalyse degradation of the anionic alginate in three steps: (I) removal of the negative charge on the carboxylate anion, (II) general base-catalysed abstraction of the proton on the C5 and (III) β-elimination of the 4-O-glycosidic bond <cite>Gacesa1986</cite>.
+
Alginate lyases (Alys) of all families, PL5-7, PL14-15, PL17-18, catalyze the depolymerization of alginate in three steps: (I) removal of the negative charge on the carboxylate anion, (II) general base-catalyzed abstraction of the proton on the C5 and (III) β-elimination of the 4-O-glycosidic bond <cite>Gacesa1986</cite>. Most PL7s are endo-active, i.e. acting within a poly- or oligosaccharide and releasing smaller alginate fragments. Some PL7 are exo-acting cleaving a monosaccharide from the non-reducing end of the polymer <cite>Thomas2013</cite>. In both modes of action, a new non-reducing end with a 4-deoxy-L-erythro-hex-4-en pyranosyl uronate residue (Δ) is formed.
Most PL7s are endo-active, i.e. acting within a poly- or oligosaccharide and releasing smaller alginate fragments, while exo-acting PL7s are cleaving a monosaccharide from the polymer termini <cite>Thomas2013</cite>. In both modes of action, a new non-reducing end with a 4-deoxy-L-erythro-hex-4 en pyranosyl uronate residue (Δ) is formed. In contrast to terrestrial PL7, marine PLs need the divalent cation calcium for substrate recognition and binding <cite>Thomas2013</cite>. Ca<sup>2+</sup> is weakening the ionic interactions between substrate (polyanion) and PL7 (polycation) by reducing the surface density of the alginate charge and therefore increasing the enzyme activity <cite>Favorov1979</cite>. There have been also reports on no increasing and even decreasing activity in presence of any mono- or divalent (metal) cation <cite>Jadtap2014 Badur2015</cite>.
+
 
 +
Some marine PL7 alginate lyases require a calcium cation for substrate recognition and binding <cite>Thomas2013</cite>. Ca<sup>2+</sup> is weakening the ionic interactions between substrate (polyanion) and PL7 (polycation) by reducing the surface density of the alginate, charge and therefore increasing the enzyme activity <cite>Favorov1979</cite>. However, there have also been reports where mono- or divalent (metal) cations did not increase or even decreased enzymatic activity <cite>Jadtap2014 Badur2015</cite>. Therefore, it is not clear if Ca<sup>2+</sup> is really needed for recognition and / or binding or if it is rather creating a more accessible form of alginate – the formation of gel particles[n1]  which might be an easier target in the marine environment as its dissolved form.
 +
 
 +
 
  
 
== Kinetics and catalytic residues ==
 
== Kinetics and catalytic residues ==
Several structural and biochemical analyses of wild type and mutated PL7s revealed five residues forming the active site: arginine (R), glutamine (Q), histidine (H), tyrosine (Y) <cite>Preston2000 Yamasaki200 Yamasaki2005</cite>, which are assembled in three highly conserved regions: R*ELR*ML, VIIGQ(I/V)H, YFKAG*Y*Q (Figure 1) <cite>Wong2000</cite>. Osawa and colleagues assumed that in PL7 ALY-1 from ''Corynebacterium ''sp. Q117+Y195 interact near the reaction site of alginate to maintain proper orientation of the substrate, R72 interacts with alginate due to the formation of salt bridges with the carboxyl groups at the C5 and H119 acts as a base to deprotonate <cite>Osawa2015</cite>.
+
Several structural and biochemical analyses of wild type and mutated PL7s revealed five residues forming the active site: arginine (R), glutamine (Q), histidine (H), tyrosine (Y) <cite>Preston2000 Yamasaki200 Yamasaki2005</cite>, which are present in three highly conserved regions: R*ELR*ML, VIIGQ(I/V)H, YFKAG*Y*Q respectively (Figure 1) <cite>Wong2000</cite>. Osawa and colleagues proposed that in PL7 ALY-1 from Corynebacterium sp. Q117+Y195 interact near the reaction site of alginate to maintain proper orientation of the substrate, R72 interacts with alginate due to the formation of salt bridges with the carboxyl groups at the C5 and H119 acts as a base to deprotonate <cite>Osawa2015</cite>. However, there can also be additional charged residues at the active site, which promote substrate recognition and binding <cite>Thomas2013</cite>. Such residues can be found in the N-terminal R*ELREML and VIIGQIH regions. Both highly conserved regions are mainly characterized by hydrophobic amino acids (especially aromatic amino acids) such as leucine, tryptophan and methionine as well as residues with planar polar side chains (especially amino acids with charged side chains) such as arginine, glutamic acid, glutamine (Figure 2). These residues have been suggested to be substrate-binding molecules <cite>Wong2000</cite>.
However, there can also be additional charged residues at the active site, which promote substrate recognition and binding <cite>Thomas2013</cite>. Such residues can be found in the N-terminal R*ELREML and VIIGQIH regions. Both highly conserved regions are mainly characterized by hydrophobic amino acids (especially aromatic amino acids) such as leucine, tryptophan and methionine as well as residues with planar polar side chains (especially amino acids with charged side chains) such as arginine, glutamic acid, glutamine (Figure 2). These residues have been suggested to be substrate-binding molecules <cite>Wong2000</cite>.
 
 
 
 
== Substrate specificities ==
 
== Substrate specificities ==
[[Image:PL7SF_Thomasetal2013.JPG|thumb|400px|'''Figure 2. Subfamilies of PL7s''' <cite>Thomas2013</cite>. Unrooted tree with bootstrap values after maximum likelihood analysis. Number refer to Uniprot accession numbers. Red dots indicate enzymes from Z. galactanivorans. Pink triangles indicate enzymes characterized biochemically. Blue squares indicate that the structure of the protein has been solved.]]
+
[[Image:PL7SF_Thomasetal2013.JPG|thumb|400px|'''Figure 2. Subfamilies of PL7s''' <cite>Thomas2013</cite>. Unrooted tree with bootstrap values after maximum likelihood analysis. Number refer to Uniprot accession numbers. Red dots indicate enzymes from ''Z. galactanivorans''. Pink triangles indicate enzymes characterized biochemically. Blue squares indicate that the structure of the protein has been solved.]]
Polysaccharide lyase family 7 (PL7) contains five subfamilies (SF) based on their sequence similarities <cite>Lombard2010</cite>, plus a so far uncharacterized sixth subfamily, which consist only of marine representatives of the Flavobacteriaceae (Figure 2) <cite>Thomas2013</cite>.  All characterized PL7 enzymes are alginate lyases specific for the anionic, gel forming polysaccharide alginate. The substrate specificity depends on the source of alginate, i.e. derived from brown seaweed or mucoid bacteria ''Pseudomonas ''spp. and ''Azotobacter vinelandii'', as well as geographical and saisonal parameters. Alginate is an heteropolysaccharide, consisting of β-D-mannuronate (M) and α-L-guluronate (G). These monosaccharides can occur in homogenous and heterogenous blocks. Hence, PL7 lyases can be mannuronate (EC 4.2.2.3), guluronate (EC 4.2.2.11) or mixed link (EC 4.2.2.-) specific. Despite the preference for M- or G-enriched blocks, most PL7s also have a moderate to low processivity for the other building block <cite>Thomas2013 Badur2015 Sim2017</cite>. PolyG specific PL7s have been found in the SF3 and SF5 <cite>Thomas2013</cite> with QIH in the second highly conserved region, while polyM specific PL7s are characterised by QVH <cite>Zhu2015 Deng2014</cite>.
+
Polysaccharide lyase family 7 (PL7) contains five subfamilies (SF) based on their sequence similarities <cite>Lombard2010</cite>, plus a so far uncharacterized sixth subfamily, which consist only of marine representatives of the Flavobacteriaceae (Figure 2) <cite>Thomas2013</cite>. The substrate specificity depends on the source of alginate, i.e. derived from brown seaweed or mucoid bacteria Pseudomonas spp. and Azotobacter vinelandii, as well as geographical and seasonal parameters. Alginate is a heteropolysaccharide, consisting of β-D-mannuronate (M) and α-L-guluronate (G). These monosaccharides can occur in homogenous and heterogenous blocks. In addition, bacterial alginate is often acetylated at the C2 and / or C3 of mannuronate, thereby shielding the substrate at this position for catalytic activity of Aly. Hence, PL7 lyases can be mannuronate (EC 4.2.2.3), guluronate (EC 4.2.2.11) or mixed link (EC 4.2.2.-) specific. Despite the preference for M- or G-enriched blocks, most PL7 also have a low to moderate activity for the other building block <cite>Thomas2013 Badur2015 Sim2017</cite>. PolyG specific PL7 have been found in the SF3 and SF5 <cite>Thomas2013</cite> with QIH in the second highly conserved region, while polyM specific PL7s are characterized by QVH <cite>Zhu2015 Deng2014</cite>.  
 
 
 
== Three-dimensional structures ==
 
== Three-dimensional structures ==
 
[[Image:EndovsexoPL7_Thomas_et_al_2013.jpg|thumb|400px|'''Figure 3. 3D Structure of endo- and exo-active PL7s''' <cite>Thomas2013</cite>. (A,B) endo AlyA1 and (C, D) exo AlyA5 from ''Zobellia galaactinovorans'' DsijT shown as cartoon (A,C) and surface structure (B,D) with superimposed tetrasaccharide from [{{PDBlink}}2ZAA PDB ID 2ZAA]. The image was conducted in PyMOL <cite>DeLano2002</cite>.]]
 
[[Image:EndovsexoPL7_Thomas_et_al_2013.jpg|thumb|400px|'''Figure 3. 3D Structure of endo- and exo-active PL7s''' <cite>Thomas2013</cite>. (A,B) endo AlyA1 and (C, D) exo AlyA5 from ''Zobellia galaactinovorans'' DsijT shown as cartoon (A,C) and surface structure (B,D) with superimposed tetrasaccharide from [{{PDBlink}}2ZAA PDB ID 2ZAA]. The image was conducted in PyMOL <cite>DeLano2002</cite>.]]
The first structure of a PL7 was determined from ''Pseudomonas aeruginosa'' by multiple isomorphous replacement (MIR) at 2.0 Å resolution <cite>Yamasaki2004</cite>. Like PL14, PL7 belongs to the jelly roll family with a wide open cleft harboring the active site (Figure 3A, B). Til date, there is only one known exoactive PL7 structure <cite>Thomas2013</cite>. ''Zobellia galaactinovorans'' DsijT is harboring, among others, two PL7 with two completely different activity modifs. AlyA1 belongs to SF3 and is an endo-active PL7. AlyA5 on the other hand belongs to SF5 and is exo-active, which active site is close by three additional loops forming a small pocket (Figure 3C, D).
+
The first structure of a PL7 was determined from Pseudomonas aeruginosa by multiple isomorphous replacement (MIR) at 2.0 Å resolution <cite>Yamasaki2004</cite>. Like PL14, PL7 belongs to the jelly roll fold with a wide open cleft harboring the active site (Figure 3A, B). Zobellia galactanivorans DsijT possess, among others, two PL7 with different modes of activity <cite>Thomas2013</cite>. AlyA1 is an endo-acting PL7 belonging to SF3, whereas AlyA5 belongs to SF5 and is exo-active which active site is closed by three loops forming a small pocket (Figure 3C, D). The highly conserved 9-amino-acid-block YFKAGVY*Q (where * is a variable residue) at the C-terminus of PL7s (Figure 2) has also been found for an extracellular pectate lyase (PL1, PDB: 1AIR) in E. chrysanthemi. Alginate and pectate/pectin lyases share the β-elimination mechanism, the recognition of substrates of a similar structure, Ca<sup>2+</sup>-binding site and primary sequence similarity, indicating that they probably possess a similar core structural fold probably important to maintain a stable 3D-confirmation <cite>Wong2000</cite>. Several alginate lyases have been reported to be multimodular enzymes with putative non-catalytic, carbohydrate-binding modules (CBMs). The first biochemically characterized CBM of an endo PL7 was a N-terminal CBM13 from Agarivorans sp. L11, which increased its substrate binding and therefore catalytic efficiency, influenced substrate specificity, the product profile and thermo stability <cite>Li2015</cite>. Contradictory observations regarding catalytic efficiency and substrate specificity were made for an endo PL7 from Vibrio splendidus OU02 DNA with an N-terminal CBM32 linked by a unique alpha-helix linker. The CBM and linker were proposed to serve as a "pivot point" somehow just pushing the product profile towards trisaccharides <cite>Lyu2018</cite>. The PL7 from Persicobacter sp. CCB-QB2 consists of three domains - a N-terminal CBM16 with unknown function, a CBM32 and the C-terminal PL7 <cite>Sim2017</cite>. Biochemical and structural analyses of full length and truncated enzyme versions have shown that (i) CBM32 and PL7 do not interact with each other and CBM32 is not enhancing the catalytic activity, (ii) CBM32 is not binding to intact alginate but to the unsaturated sugar at the non-reducing end of cleaved alginate, (iii) CBM32 and PL7 both possess a Ca<sup>2+</sup>- and K<sup>+</sup>-binding site and conserved residues probably binding to carboxyl groups of negatively charged carbohydrates.  
The highly conserved 9-amino-acid-block YFKAGVY*Q (where * is a variable residue) at the C-terminus of PL7s (Figure 2) has also been found for an extracellular pectate lyase in ''E. chrysanthemi'' (Keen & Tamaki, 1986)            . Alginate and pectate / pectin lyases share several features such as β-elimination, the recognition of substrates of a similar structure and primary sequence similarity, indicating that they probably share a similar core structural fold. Since alginate lyases and pectinases differ in substrate specificity, it is likely not related to substrate recognition, but rather to maintaining a stable 3D-conformation <cite>Wong2000</cite>.
 
Several alginate lyases have been reported to be multimodular domain enzymes with putative non-catalytic, carbohydrate-binding modules (CBMs). However, their exact role has been barely investigated yet. The first biochemically characterized CBM of an endo PL7 was a N-terminal CBM13 from ''Agarivorans'' sp. L11, which not only increased its substrate binding ability and therefore catalytic efficiency, but also substrate preference and product profiling as well as thermostability <cite>Li2015</cite>. Contradictory observations regarding catalytic effiency and substrate specificity were made for an endo PL7 from ''Vibrio splendidus'' OU02 DNA with an N-terminal CBM32 linked by a unique alpha helix linker <cite>Lyu2018</cite>. Nevertheless, the CBM and und linker were supposed to serve as "pivont point" affecting the product distribution towards trisaccharides. The PL7 from ''Persicobacter'' sp. CCB-QB2 is even consisting of three domains - a N-terminal CBM16 with still unclear function, C-terminal catalytic domain and a  CBM32, which is located between both domains <cite>Sim2017</cite>. This CBM32 is also not enhancing the catalytic activity and is not binding alginate, but the cleaved termini during catalysis. The crystal structures revealed an arginine residue which is possibly binding to the carboxylic group and a conserved Ca<sup>2+</sup> binding site being most likely essential for the maintenance of the overall fold.
 
 
 
  
 
== Gene transfer of Alys among different habitats ==
 
== Gene transfer of Alys among different habitats ==
Alginate degrading organisms often posses specified gene clusters for glycan utilization which contain, among other proteins such as transporters, several endo- and exo-actining Alys of different families. These gene clusters are called polysaccharide utilization loci (PULs) for Bacteriodetes <cite>Sonnenber2010</cite> or alginolytic operons in case a SusCD pair transporter is missing or replaced by a different transporter system. It has been shown that these gene cluster can be transfered horizontally from one organism to another and thereby even cross different environmental habitats <cite>Hehemann2010</cite>. The first alginate utilization system (AUS) was found in ''Zobellia galactinovorans'' which contains two clusters harboring five out of seven Alys (3 PL7s). Those operons originated from an ancestral marine Flavobacterium and were independently transferred to marine ''Proteobacteria ''and Japanese gut Bacteriodetes by lateral gene transfer (LGT) <cite>Thomas2012</cite>.
+
Alginate degrading organisms often posse specified gene clusters for glycan utilization which contain, among other proteins such as transporters, several endo- and exo-acting Alys of different families. These gene clusters are called polysaccharide utilization loci (PULs) for Bacteriodetes <cite>Sonnenber2010</cite> or alginolytic operons in case a SusCD pair transporter is missing or replaced by a different transporter system. It has been shown that these gene clusters can be transferred horizontally from one organism to another and thereby even cross different environmental habitats <cite>Hehemann2010</cite>. The first alginate utilization system (AUS) was found in Zobellia galactinovorans which contains two clusters harboring five out of seven Alys (3x PL7). Those operons originated from an ancestral marine Flavobacterium and were independently transferred to marine Proteobacteria and Japanese gut Bacteriodetes by lateral gene transfer (LGT) <cite>Thomas2012</cite>.
  
 +
A more detailed studied on horizontal gene transfer of PL7 between marine, ecophysiological different Vibrionaceae isolates revealed rapid adaptation of closely related but speciated bacterial populations, resulting into a fine-scale of resource partitioning. The exchange of PL7 between marine microbes drove the evolution of polysaccharide degrading pathways, which might have led to three ecotypes – the pioneers, which degrade the polymer into oligomers, the harvester (intermediate of the other two types) and the scavenger, which can only utilize very small oligos created by the pioneers <cite>Hehemann2016</cite>.
  
 
== Family Firsts ==
 
== Family Firsts ==
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#Jagtap2014 pmid=24795372
 
#Jagtap2014 pmid=24795372
 
#Badur2015 pmid=25556193
 
#Badur2015 pmid=25556193
 +
#Hehemann2016 pmid=27653556
 
</biblio>
 
</biblio>
 
[[Category:Polysaccharide Lyase Families|PL007]]
 
[[Category:Polysaccharide Lyase Families|PL007]]

Revision as of 02:06, 4 November 2019

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Polysaccharide Lyase Family PL7
3D Structure β jelly roll
Mechanism β-elimination
Active site residues R, Q, H, 2xY
CAZy DB link
http://www.cazy.org/PL7.html


Mechanism

Figure 1. Multiple protein sequence alignment of Aly PL7 as well as a secondary structure prediction with the crystallized PL7 from Klabsiella pneumoniae (PDB ID 4OZX). Conserved residues in the homologues are colored in red and (putative) catalytic residues are indicated by a star. The multiple protein sequence alignment was done with Espript3.0 [1].

Alginate lyases (Alys) of all families, PL5-7, PL14-15, PL17-18, catalyze the depolymerization of alginate in three steps: (I) removal of the negative charge on the carboxylate anion, (II) general base-catalyzed abstraction of the proton on the C5 and (III) β-elimination of the 4-O-glycosidic bond [2]. Most PL7s are endo-active, i.e. acting within a poly- or oligosaccharide and releasing smaller alginate fragments. Some PL7 are exo-acting cleaving a monosaccharide from the non-reducing end of the polymer [3]. In both modes of action, a new non-reducing end with a 4-deoxy-L-erythro-hex-4-en pyranosyl uronate residue (Δ) is formed.

Some marine PL7 alginate lyases require a calcium cation for substrate recognition and binding [3]. Ca2+ is weakening the ionic interactions between substrate (polyanion) and PL7 (polycation) by reducing the surface density of the alginate, charge and therefore increasing the enzyme activity [4]. However, there have also been reports where mono- or divalent (metal) cations did not increase or even decreased enzymatic activity [5, 6]. Therefore, it is not clear if Ca2+ is really needed for recognition and / or binding or if it is rather creating a more accessible form of alginate – the formation of gel particles[n1] which might be an easier target in the marine environment as its dissolved form.


Kinetics and catalytic residues

Several structural and biochemical analyses of wild type and mutated PL7s revealed five residues forming the active site: arginine (R), glutamine (Q), histidine (H), tyrosine (Y) [7, 8, 9], which are present in three highly conserved regions: R*ELR*ML, VIIGQ(I/V)H, YFKAG*Y*Q respectively (Figure 1) [10]. Osawa and colleagues proposed that in PL7 ALY-1 from Corynebacterium sp. Q117+Y195 interact near the reaction site of alginate to maintain proper orientation of the substrate, R72 interacts with alginate due to the formation of salt bridges with the carboxyl groups at the C5 and H119 acts as a base to deprotonate [11]. However, there can also be additional charged residues at the active site, which promote substrate recognition and binding [3]. Such residues can be found in the N-terminal R*ELREML and VIIGQIH regions. Both highly conserved regions are mainly characterized by hydrophobic amino acids (especially aromatic amino acids) such as leucine, tryptophan and methionine as well as residues with planar polar side chains (especially amino acids with charged side chains) such as arginine, glutamic acid, glutamine (Figure 2). These residues have been suggested to be substrate-binding molecules [10].

Substrate specificities

Figure 2. Subfamilies of PL7s [3]. Unrooted tree with bootstrap values after maximum likelihood analysis. Number refer to Uniprot accession numbers. Red dots indicate enzymes from Z. galactanivorans. Pink triangles indicate enzymes characterized biochemically. Blue squares indicate that the structure of the protein has been solved.

Polysaccharide lyase family 7 (PL7) contains five subfamilies (SF) based on their sequence similarities [12], plus a so far uncharacterized sixth subfamily, which consist only of marine representatives of the Flavobacteriaceae (Figure 2) [3]. The substrate specificity depends on the source of alginate, i.e. derived from brown seaweed or mucoid bacteria Pseudomonas spp. and Azotobacter vinelandii, as well as geographical and seasonal parameters. Alginate is a heteropolysaccharide, consisting of β-D-mannuronate (M) and α-L-guluronate (G). These monosaccharides can occur in homogenous and heterogenous blocks. In addition, bacterial alginate is often acetylated at the C2 and / or C3 of mannuronate, thereby shielding the substrate at this position for catalytic activity of Aly. Hence, PL7 lyases can be mannuronate (EC 4.2.2.3), guluronate (EC 4.2.2.11) or mixed link (EC 4.2.2.-) specific. Despite the preference for M- or G-enriched blocks, most PL7 also have a low to moderate activity for the other building block [3, 6, 13]. PolyG specific PL7 have been found in the SF3 and SF5 [3] with QIH in the second highly conserved region, while polyM specific PL7s are characterized by QVH [14, 15].

Three-dimensional structures

Figure 3. 3D Structure of endo- and exo-active PL7s [3]. (A,B) endo AlyA1 and (C, D) exo AlyA5 from Zobellia galaactinovorans DsijT shown as cartoon (A,C) and surface structure (B,D) with superimposed tetrasaccharide from PDB ID 2ZAA. The image was conducted in PyMOL [16].

The first structure of a PL7 was determined from Pseudomonas aeruginosa by multiple isomorphous replacement (MIR) at 2.0 Å resolution [17]. Like PL14, PL7 belongs to the jelly roll fold with a wide open cleft harboring the active site (Figure 3A, B). Zobellia galactanivorans DsijT possess, among others, two PL7 with different modes of activity [3]. AlyA1 is an endo-acting PL7 belonging to SF3, whereas AlyA5 belongs to SF5 and is exo-active which active site is closed by three loops forming a small pocket (Figure 3C, D). The highly conserved 9-amino-acid-block YFKAGVY*Q (where * is a variable residue) at the C-terminus of PL7s (Figure 2) has also been found for an extracellular pectate lyase (PL1, PDB: 1AIR) in E. chrysanthemi. Alginate and pectate/pectin lyases share the β-elimination mechanism, the recognition of substrates of a similar structure, Ca2+-binding site and primary sequence similarity, indicating that they probably possess a similar core structural fold probably important to maintain a stable 3D-confirmation [10]. Several alginate lyases have been reported to be multimodular enzymes with putative non-catalytic, carbohydrate-binding modules (CBMs). The first biochemically characterized CBM of an endo PL7 was a N-terminal CBM13 from Agarivorans sp. L11, which increased its substrate binding and therefore catalytic efficiency, influenced substrate specificity, the product profile and thermo stability [18]. Contradictory observations regarding catalytic efficiency and substrate specificity were made for an endo PL7 from Vibrio splendidus OU02 DNA with an N-terminal CBM32 linked by a unique alpha-helix linker. The CBM and linker were proposed to serve as a "pivot point" somehow just pushing the product profile towards trisaccharides [19]. The PL7 from Persicobacter sp. CCB-QB2 consists of three domains - a N-terminal CBM16 with unknown function, a CBM32 and the C-terminal PL7 [13]. Biochemical and structural analyses of full length and truncated enzyme versions have shown that (i) CBM32 and PL7 do not interact with each other and CBM32 is not enhancing the catalytic activity, (ii) CBM32 is not binding to intact alginate but to the unsaturated sugar at the non-reducing end of cleaved alginate, (iii) CBM32 and PL7 both possess a Ca2+- and K+-binding site and conserved residues probably binding to carboxyl groups of negatively charged carbohydrates.

Gene transfer of Alys among different habitats

Alginate degrading organisms often posse specified gene clusters for glycan utilization which contain, among other proteins such as transporters, several endo- and exo-acting Alys of different families. These gene clusters are called polysaccharide utilization loci (PULs) for Bacteriodetes [20] or alginolytic operons in case a SusCD pair transporter is missing or replaced by a different transporter system. It has been shown that these gene clusters can be transferred horizontally from one organism to another and thereby even cross different environmental habitats [21]. The first alginate utilization system (AUS) was found in Zobellia galactinovorans which contains two clusters harboring five out of seven Alys (3x PL7). Those operons originated from an ancestral marine Flavobacterium and were independently transferred to marine Proteobacteria and Japanese gut Bacteriodetes by lateral gene transfer (LGT) [22].

A more detailed studied on horizontal gene transfer of PL7 between marine, ecophysiological different Vibrionaceae isolates revealed rapid adaptation of closely related but speciated bacterial populations, resulting into a fine-scale of resource partitioning. The exchange of PL7 between marine microbes drove the evolution of polysaccharide degrading pathways, which might have led to three ecotypes – the pioneers, which degrade the polymer into oligomers, the harvester (intermediate of the other two types) and the scavenger, which can only utilize very small oligos created by the pioneers [23].

Family Firsts

First catalytic endo-activity
polyM PL7 from Photobacterium ATCC 433367 [24], polyG PL7 from Klebsiella pneumoniae subbsp. aerogenes [25]
First catalytic exo-activity
AlyA5 from Zobellia galactanivorans DsijT [3]
First 3-D apo-structure
PA1167 from Pseudomonas aeruginosa [17]
First 3-D holo-structure
A1-II from Sphingomons sp. A1 [26]
First characterised CBM
AlyL2 containing a N-terminal CBM13 from Agarivorans sp. L11 [18]


References

  1. Robert X and Gouet P. (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320-4. DOI:10.1093/nar/gku316 | PubMed ID:24753421 | HubMed [Robert2014]
  2. Gacesa P. (1987) Alginate‐modifying enzymes: A proposed unified mechanism of action for the lyases and epimerases. FEBS Letters, 212, 1873-3468. DOI:10.1016/0014-5793(87)81344-3
    [Gacesa1986]
  3. Thomas F, Lundqvist LC, Jam M, Jeudy A, Barbeyron T, Sandström C, Michel G, and Czjzek M. (2013) Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct modes of action and exquisite adaptation to their natural substrate. J Biol Chem. 288, 23021-37. DOI:10.1074/jbc.M113.467217 | PubMed ID:23782694 | HubMed [Thomas2013]
  4. Favorov VV, Vozhova EI, Denisenko VA, and Elyakova LA. (1979) A study of the reaction catalysed by alginate lyase VI from the sea mollusc, Littorina sp. Biochim Biophys Acta. 569, 259-66. DOI:10.1016/0005-2744(79)90061-5 | PubMed ID:476128 | HubMed [Favorov1979]
  5. Badur AH, Jagtap SS, Yalamanchili G, Lee JK, Zhao H, and Rao CV. (2015) Alginate lyases from alginate-degrading Vibrio splendidus 12B01 are endolytic. Appl Environ Microbiol. 81, 1865-73. DOI:10.1128/AEM.03460-14 | PubMed ID:25556193 | HubMed [Badur2015]
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