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John T. Jones - One of the best experts on this subject based on the ideXlab platform.
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cloning and characterization of pectate Lyases expressed in the esophageal gland of the pine wood nematode bursaphelenchus xylophilus
Molecular Plant-microbe Interactions, 2006Co-Authors: Taisei Kikuchi, Hajime Shibuya, Takuya Aikawa, John T. JonesAbstract:Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xylophilus. The deduced amino acid sequences of these pectate Lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55 degrees C and pH 8 to 10 like other pectate Lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate Lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate Lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.
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Cloning and Characterization of Pectate Lyases Expressed in the Esophageal Gland of the Pine Wood Nematode Bursaphelenchus xylophilus
Molecular Plant-Microbe Interactions®, 2006Co-Authors: Taisei Kikuchi, Hajime Shibuya, Takuya Aikawa, John T. JonesAbstract:Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xy-lophilus. The deduced amino acid sequences of these pectate Lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55°C and pH 8 to 10 like other pectate Lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate Lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate Lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.
Zhong Yao - One of the best experts on this subject based on the ideXlab platform.
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Construction and biochemical characterization of a novel hybrid alginate lyase with high activity by module recombination to prepare alginate oligosaccharides
International Journal of Biological Macromolecules, 2020Co-Authors: Shengsheng Cao, Benwei Zhu, Zhong YaoAbstract:Abstract Alginate Lyases are essential tools to prepare alginate oligosaccharides with various biological activities. However, alginate Lyases with excellent properties such as high activity and good thermal stability are still in shortage. Therefore, it is crucial to exploit new alginate Lyases with high activity and polysaccharide-degrading efficiency for alginate oligosaccharide preparation. Herein, we proposed to construct a novel hybrid alginate lyase with improved property by module recombination. The hybrid alginate lyase, designated as Aly7C, was successfully constructed by recombining the carbohydrate binding module (CBM) of Aly7A with the catalytic module of Aly7B. Interestingly, the hybrid enzyme Aly7C exhibited higher activity than the catalytic domain. Moreover, it could degrade sodium alginate, polyM and polyG into oligosaccharides with degrees of polymerization (Dps) 2–5, which exhibit perfect product specificity. This work provides a new insight into well-defined generation of alginate oligosaccharides with associated CBMs and enhances the understanding of functions of the modules.
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Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides.
International Journal of Biological Macromolecules, 2020Co-Authors: Mingyang Wang, Benwei Zhu, Zhong YaoAbstract:Abstract Alginate Lyases are important enzymes to prepare alginate oligosaccharides for industrial applications and elucidating the degradation pattern of alginate Lyases is essential for expanding their applications. Herein, we cloned and expressed AlyPL6, a novel member of polysaccharide lyase family-6 (PL6) with high activity from Pedobacter hainanensis NJ-02. It was found that AlyPL6 could recognize tetrasaccharide as the minimal substrate to release oligosaccharides with low degrees of polymerization (DPs). As a result, it could be speculated that the cleavage site of substrate is located between −1 and +1 subsites. For further application, AlyPL6 was then immobilized onto mesoporous titanium oxide particles (MTOPs) with >55.4% of maximal activity retained at 45 °C after it was reused for 10 times. The research provided extended insights into the substrate recognition and degradation pattern of PL 6 alginate Lyases, which may benefit the further application of alginate Lyases a lot.
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elucidation of degrading pattern and substrate recognition of a novel bifunctional alginate lyase from flammeovirga sp nj 04 and its use for preparation alginate oligosaccharides
Biotechnology for Biofuels, 2019Co-Authors: Benwei Zhu, Yun Sun, Limin Ning, Zhong YaoAbstract:The alginate oligosaccharides have been widely used in agriculture, medicine, and food industries due to their versatile physiological functions such as antioxidant, anticoagulant, and antineoplastic activities. The bifunctional alginate Lyases can degrade the alginate polysaccharide more efficiently into alginate oligosaccharides. Therefore, it is crucial to discover new bifunctional alginate lyase for alginate oligosaccharide production. Herein, a novel bifunctional alginate lyase FsAlgB was cloned and identified from deep-sea bacterium Flammeovirga sp. NJ-04, which exhibited broad substrate specificity and the highest activity (1760.8 U/mg) at pH 8.0 and 40 °C. Furthermore, the Km values of FsAlgB towards polyG (0.69 mM) and polyMG (0.92 mM) were lower than that towards sodium alginate (1.28 mM) and polyM (2.06 mM). Recombinant FsAlgB was further characterized as an endolytic alginate lyase, and it can recognize the tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of − 3 and + 1. This study provided extended insights into the substrate recognition and degrading pattern of alginate Lyases with broad substrate specificity.
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Elucidation of degrading pattern and substrate recognition of a novel bifunctional alginate lyase from Flammeovirga sp. NJ-04 and its use for preparation alginate oligosaccharides
BMC, 2019Co-Authors: Benwei Zhu, Yun Sun, Limin Ning, Zhong YaoAbstract:Abstract Background The alginate oligosaccharides have been widely used in agriculture, medicine, and food industries due to their versatile physiological functions such as antioxidant, anticoagulant, and antineoplastic activities. The bifunctional alginate Lyases can degrade the alginate polysaccharide more efficiently into alginate oligosaccharides. Therefore, it is crucial to discover new bifunctional alginate lyase for alginate oligosaccharide production. Results Herein, a novel bifunctional alginate lyase FsAlgB was cloned and identified from deep-sea bacterium Flammeovirga sp. NJ-04, which exhibited broad substrate specificity and the highest activity (1760.8 U/mg) at pH 8.0 and 40 °C. Furthermore, the K m values of FsAlgB towards polyG (0.69 mM) and polyMG (0.92 mM) were lower than that towards sodium alginate (1.28 mM) and polyM (2.06 mM). Recombinant FsAlgB was further characterized as an endolytic alginate lyase, and it can recognize the tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of − 3 and + 1. Conclusion This study provided extended insights into the substrate recognition and degrading pattern of alginate Lyases with broad substrate specificity
Yuzhong Zhang - One of the best experts on this subject based on the ideXlab platform.
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Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the Arctic.
Journal of Biological Chemistry, 2020Co-Authors: Xiulan Chen, Yuzhong Zhang, Fang Dong, Yin Chen, Xiao-hui Sun, Haitao Ding, Peng WangAbstract:Alginate Lyases play important roles in alginate degradation in the ocean. Although a large number of alginate Lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism, that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule, and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg82 and Tyr190 at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate, and that His127, Tyr244, Arg78, and Gln125 mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase, and demonstrate that such residues involved in alginate positioning are conserved in other alginate Lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate Lyases.
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alginate lyase aly36b is a new bacterial member of the polysaccharide lyase family 36 and catalyzes by a novel mechanism with lysine as both the catalytic base and catalytic acid
Journal of Molecular Biology, 2019Co-Authors: Fang Dong, Xiulan Chen, Yuzhong Zhang, Peng Wang, Yin ChenAbstract:Alginate Lyases, which are important in both basic and applied sciences, fall into ten polysaccharide lyase (PL) families. PL36 is a newly established family that includes 39 bacterial sequences and one eukaryotic sequence. Till now, the structures or catalytic mechanisms of PL36 alginate Lyases have yet to be revealed. Here, we characterized a novel PL36 alginate lyase, Aly36B, from Chitinophaga sp. MD30. Aly36B is a polymannuronate specific endolytic alginate lyase. To probe the catalytic mechanism of Aly36B, the structures of wild-type Aly36B and its mutants (K143A/Y185A in complex with alginate tetrasaccharide and K143A/M171A with trisaccharide) were solved. The overall structure of Aly36B belongs to the β-jelly roll scaffold, adopting a typical β-sandwich fold. Aly36B contains a Ca2+, which is far away from the active center and plays an important role in stabilizing the structure of Aly36B. Based on structural and mutational analyses, the catalytic mechanism of Aly36B for alginate degradation was explained. During catalysis, Arg169, Tyr185, and Tyr187 are responsible for neutralizing the negative charge of the substrate, and Lys143 acts as both the catalytic base and the catalytic acid, which represents a new kind of catalytic mechanism of alginate Lyases. Sequence alignment shows that these four residues involved in catalysis are highly conserved in all PL36 sequences, suggesting that PL36 alginate Lyases may adopt a similar catalytic mechanism. Taken together, this study reveals the molecular structure and catalytic mechanism of a PL36 alginate lyase, broadening our knowledge on alginate Lyases and facilitating future biotechnological applications of PL36 alginate Lyases.
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novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase alygc from polysaccharide lyase family 6
Journal of Biological Chemistry, 2017Co-Authors: Fang Dong, Yuzhong Zhang, Peng Wang, Haiyan Cao, Xiuhua Pang, Xiulan ChenAbstract:Alginate Lyases that degrade alginate via a β-elimination reaction fall into seven polysaccharide lyase (PL) families. Although the structures and catalytic mechanisms of alginate Lyases in the other PL families have been clarified, those in family PL6 have yet to be revealed. Here, the crystal structure of AlyGC, a PL6 alginate lyase from marine bacterium Glaciecola chathamensis S18K6T, was solved, and its catalytic mechanism was illustrated. AlyGC is a homodimeric enzyme and adopts a structure distinct from other alginate Lyases. Each monomer contains a catalytic N-terminal domain and a functionally unknown C-terminal domain. A combined structural and mutational analysis using the structures of AlyGC and of an inactive mutant R241A in complex with an alginate tetrasaccharide indicates that conformational changes occur in AlyGC when a substrate is bound and that the two active centers in AlyGC may not bind substrates simultaneously. The C-terminal domain is shown to be essential for the dimerization and the catalytic activity of AlyGC. Residues Tyr130, Arg187, His242, Arg265, and Tyr304 in the active center are also important for the activity of AlyGC. In catalysis, Lys220 and Arg241 function as the Bronsted base and acid, respectively, and a Ca2+ in the active center neutralizes the negative charge of the C5 carboxyl group of the substrate. Finally, based on our data, we propose a metal ion-assisted catalytic mechanism of AlyGC for alginate cleavage with a state change mode, which provides a better understanding for polysaccharide Lyases and alginate degradation.
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characterization of a new cold adapted and salt activated polysaccharide lyase family 7 alginate lyase from pseudoalteromonas sp sm0524
Frontiers in Microbiology, 2016Co-Authors: Xiulan Chen, Sheng Dong, Xiying Zhang, Yuzhong Zhang, Fang Dong, Baicheng Zhou, Binbin XieAbstract:Marine bacterial alginate Lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate Lyases have been characterized, reports on alginate Lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate Lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30oC and remained 19% of the highest activity at 5oC. AlyPM was unstable at temperatures above 30oC and had a low Tm of 37oC. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5-1.2 M NaCl was 6-fold higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20 folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a novel PL7 endo-alginate lyase with special characteristics.
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molecular insight into the role of the n terminal extension in the maturation substrate recognition and catalysis of a bacterial alginate lyase from polysaccharide lyase family 18
Journal of Biological Chemistry, 2014Co-Authors: Sheng Dong, Xiying Zhang, Xiulan Chen, Peng Wang, Xiuhua Pang, Baicheng Zhou, Binbin Xie, Tiandi Wei, Qilong Qin, Yuzhong ZhangAbstract:Bacterial alginate Lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate Lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a β-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr(353) may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate Lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.
Takao Ojima - One of the best experts on this subject based on the ideXlab platform.
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discovery of a novel alginate lyase from nitratiruptor sp sb155 2 thriving at deep sea hydrothermal vents and identification of the residues responsible for its heat stability
Journal of Biological Chemistry, 2016Co-Authors: Akira Inoue, Moe Anraku, Satoshi Nakagawa, Takao OjimaAbstract:Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of ϵ-Proteobacteria Nitratiruptor sp. SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate Lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 °C and around 6, respectively. A high concentration of NaCl (0.8-1.4 m) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 °C for 30 min. Heat stability was decreased in the presence of 5 mm DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate Lyases.
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Characterization of an Eukaryotic PL-7 Alginate Lyase in the Marine Red Alga Pyropia yezoensis
Current Biotechnology, 2015Co-Authors: Akira Inoue, Chieco Mashino, Toshiki Uji, Naotsune Saga, Koji Mikami, Takao OjimaAbstract:Background Alginate Lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate Lyases. However, all characterized PL-7 alginate Lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis. Methods The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated. Results PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate Lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage. Conclusion This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.
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cdna cloning and bacterial expression of a pl 14 alginate lyase from a herbivorous marine snail littorina brevicula
Carbohydrate Research, 2012Co-Authors: Mohammad Matiur Rahman, Akira Inoue, Ling Wang, Takao OjimaAbstract:Abstract Herbivorous marine snails like Littorina species are known to possess alginate Lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) Lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5′- and 3′-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43–53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28 . Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate Lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.
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Heat-stability and primary structure of the major alginate lyase isozyme LbAly35 from Littorina brevicula
Fisheries Science, 2012Co-Authors: Ling Wang, Mohammad Matiur Rahman, Akira Inoue, Takao OjimaAbstract:Previously we isolated the major alginate lyase isozyme LbAly35 from a marine snail Littorina brevicula and showed that this enzyme was significantly heat stable in a broad pH range compared with other molluscan alginate Lyases (Hata et al., Fish Sci 75:755–763, 2009). LbAly35 showed practically no similarity to other molluscan alginate Lyases in the N-terminal amino-acid sequence of 20 residues and no cross-reactivity with anti-abalone alginate lyase antiserum. These led us to consider that the primary structure of LbAly35 is considerably deviated from other molluscan enzymes. Thus, in the present study, we first compared the thermal stability of LbAly35 with an abalone alginate lyase, HdAly, and found that the first order inactivation rate constants for LbAly35 at 40 and 45 °C were 1/20 and 1/45 of those for HdAly, respectively. Then, we cloned cDNAs encoding LbAly35 and characterized its deduced amino-acid sequence comparing with those of other molluscan alginate Lyases. The cDNAs were amplified by polymerase chain reaction (PCR) and 5′- and 3′-RACE PCRs from the L. brevicula hepatopancreas cDNA using degenerated primers synthesized on the basis of partial amino-acid sequences of LbAly35. The cDNA covering the entire translational region of LbAly35 comprised 1,093 bp and encoded an amino-acid sequence of 296 residues. The amino-acid sequence consisted of an initiation methionine, a putative signal peptide for secretion (22 residues), a propeptide-like region (10 residues), and a mature LbAly35 domain of 263 residues. Although the N-terminal region of LbAly35 was significantly deviated from those of other molluscan alginate Lyases, the catalytic domain of LbAly35 showed ~45 % identity to other molluscan enzymes which had been classified under polysaccharide-lyase-family-14 (PL-14). In addition, the amino-acid residues crucially important for the catalytic actions of PL-14 enzymes were also conserved in LbAly35. Accordingly, LbAly35 was regarded as a member of PL-14 as other molluscan alginate Lyases despite the significant deviation of its N-terminal region.
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Comparative study on general properties of alginate Lyases from some marine gastropod mollusks
Fisheries Science, 2009Co-Authors: Mami Hata, Mohammad Matiur Rahman, Akira Inoue, Yuya Kumagai, Satoru Chiba, Hiroyuki Tanaka, Takao OjimaAbstract:Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via β-elimination, producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino acid sequences of alginate Lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate Lyases. The alginate Lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate Lyases with molecular masses of approximately 28, 34, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate Lyases with molecular masses of 35, 32, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher-order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino acid sequences, while the L. brevicula enzymes were not.
William Helbert - One of the best experts on this subject based on the ideXlab platform.
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Functional Exploration of the Polysaccharide Lyase Family PL6
PLoS ONE, 2017Co-Authors: Sophie Mathieu, Bernard Henrissat, Flavien Labre, Gudmund Skjåk-bræk, William HelbertAbstract:Alginate, the main cell-wall polysaccharide of brown algae, is composed of two residues: mannuronic acid (M-residues) and, its C5-epimer, guluronic acid (G-residues). Alginate Lyases define a class of enzymes that cleave the glycosidic bond of alginate by β-elimination. They are classified according to their ability to recognize the distribution of M-and G-residues and are named M-, G-or MG-Lyases. In the CAZy database, alginate Lyases have been grouped by sequence similarity into seven distinct polysaccharide lyase families. The polysaccharide lyase family PL6 is subdivided into three subfamilies. Subfamily PL6_1 includes three biochemically characterized enzymes (two alginate Lyases and one derma-tan sulfatase lyase). No characterized enzymes have been described in the two other sub-families (PL6_2 and PL6_3). To improve the prediction of polysaccharide-lyase activity in the PL6 family, we reexamined the classification of the PL6 family and biochemically characterized a set of enzymes reflecting the diversity of the protein sequences. Our results show that subfamily PL6_1 includes two dermatan sulfates Lyases and several alginate Lyases that have various substrate specificities and modes of action. In contrast, subfamilies PL6_2 and PL6_3 were found to contain only endo-poly-MG-Lyases.
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new family of ulvan Lyases identified in three isolates from the alteromonadales order
Journal of Biological Chemistry, 2016Co-Authors: Moran Kopel, William Helbert, Yana Belnik, Vitaliy Buravenkov, Asael Herman, Ehud BaninAbstract:Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), or d-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a β-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan Lyases from three Alteromonadales isolated bacteria. Two homologous ulvan Lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan Lyases and therefore are the first representatives of a new family of polysaccharide Lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and (1)H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently.
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ulvan Lyases isolated from the flavobacteria persicivirga ulvanivorans are the first members of a new polysaccharide lyase family
Journal of Biological Chemistry, 2011Co-Authors: Pi Nyvall Collen, Jeanfrancois Sassi, Helene Rogniaux, Helene Marfaing, William HelbertAbstract:Ulvans are complex sulfated polysaccharides found in the cell walls of green algae belonging to the genus Ulva. These polysaccharides are composed of disaccharide repetition moieties made up of sulfated rhamnose linked to either glucuronic acid, iduronic acid, or xylose. Two ulvan Lyases of 30 and 46 kDa were purified from the culture supernatant of Persicivirga ulvanivorans. Based on peptide sequencing, the gene encoding the 46-kDa ulvan lyase was cloned. Sequence analysis revealed that the protein is modular and possesses a catalytic module similar to that of the 30-kDa ulvan lyase along with a module of unknown function. The ulvan-degrading function of the gene was confirmed by expression of the catalytic module in a heterologous system. The gene encoding the catalytic module has no sequence homolog in sequence databases and is likely to be the first member of a novel polysaccharide lyase family. Analysis of degradation products showed that both the 30- and 46-kDa ulvan Lyases are endolytic and cleave the glycosidic bond between the sulfated rhamnose and a glucuronic or iduronic acid.
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ulvan Lyases isolated from the flavobacteria persicivirga ulvanivorans are the first members of a new polysaccharide
2011Co-Authors: Pi Nyvall Collen, Jeanfrancois Sassi, Helene Rogniaux, Helene Marfaing, William HelbertAbstract:. Based on peptide sequencing, the gene encoding the46-kDa ulvan lyase was cloned. Sequence analysis revealed thatthe protein is modular and possesses a catalytic module similarto that of the 30-kDa ulvan lyase along with a module ofunknown function. The ulvan-degrading function of the genewasconfirmedbyexpressionofthecatalyticmoduleinaheter-ologoussystem.Thegeneencodingthecatalyticmodulehasnosequence homolog in sequence databases and is likely to be thefirst member of a novel polysaccharide lyase family. Analysis ofdegradation products showed that both the 30- and 46-kDaulvan Lyases are endolytic and cleave the glycosidic bondbetween the sulfated rhamnose and a glucuronic or iduronicacid.
