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Yuzhong Zhang - One of the best experts on this subject based on the ideXlab platform.
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mechanistic insights into substrate recognition and catalysis of a new ulvan Lyase of the polysaccharide Lyase family 24
Applied and Environmental Microbiology, 2021Co-Authors: Fang Dong, Xiying Zhang, Yuzhong Zhang, Peng Wang, Haiyan Cao, Xiao-hui Sun, Andrew Mcminn, Xiulan ChenAbstract:Ulvan is an important marine polysaccharide. Bacterial ulvan Lyases play important roles in ulvan degradation and marine carbon cycling. Until now, only a small number of ulvan Lyases have been characterized. Here, a new ulvan Lyase, Uly1, belonging to polysaccharide Lyase family 24 (PL24) from the marine bacterium Catenovulum maritimum, is characterized. The optimal temperature and pH for Uly1 to degrade ulvan are 40°C and pH 9.0, respectively. Uly1 degrades ulvan polysaccharides in the endolytic manner, mainly producing ΔRha3S, consisting of an unsaturated 4-deoxy-l-threo-hex-4-enopyranosiduronic acid and a 3-O-sulfated α-l-rhamnose. The structure of Uly1 was resolved at a 2.10-A resolution. Uly1 adopts a seven-bladed β-propeller architecture. Structural and site-directed mutagenesis analyses indicate that four highly conserved residues, H128, H149, Y223, and R239, are essential for catalysis. H128 functions as both the catalytic acid and base, H149 and R239 function as the neutralizers, and Y223 plays a supporting role in catalysis. Structural comparison and sequence alignment suggest that Uly1 and many other PL24 enzymes may directly bind the substrate near the catalytic residues for catalysis, different from the PL24 ulvan Lyase LOR_107, which adopts a two-stage substrate binding process. This study provides new insights into ulvan Lyases and ulvan degradation. IMPORTANCE Ulvan is a major cell wall component of green algae of the genus Ulva. Many marine heterotrophic bacteria can produce extracellular ulvan Lyases to degrade ulvan for a carbon nutrient. In addition, ulvan has a range of physiological bioactivities based on its specific chemical structure. Ulvan Lyase thus plays an important role in marine carbon cycling and has great potential in biotechnological applications. However, only a small number of ulvan Lyases have been characterized over the past 10 years. Here, based on biochemical and structural analyses, a new ulvan Lyase of polysaccharide Lyase family 24 is characterized, and its substrate recognition and catalytic mechanisms are revealed. Moreover, a new substrate binding process adopted by PL24 ulvan Lyases is proposed. This study offers a better understanding of bacterial ulvan Lyases and is helpful for studying the application potentials of ulvan Lyases.
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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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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, Xiying Zhang, Yuzhong Zhang, Sheng Dong, 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.
Xiulan Chen - One of the best experts on this subject based on the ideXlab platform.
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mechanistic insights into substrate recognition and catalysis of a new ulvan Lyase of the polysaccharide Lyase family 24
Applied and Environmental Microbiology, 2021Co-Authors: Fang Dong, Xiying Zhang, Yuzhong Zhang, Peng Wang, Haiyan Cao, Xiao-hui Sun, Andrew Mcminn, Xiulan ChenAbstract:Ulvan is an important marine polysaccharide. Bacterial ulvan Lyases play important roles in ulvan degradation and marine carbon cycling. Until now, only a small number of ulvan Lyases have been characterized. Here, a new ulvan Lyase, Uly1, belonging to polysaccharide Lyase family 24 (PL24) from the marine bacterium Catenovulum maritimum, is characterized. The optimal temperature and pH for Uly1 to degrade ulvan are 40°C and pH 9.0, respectively. Uly1 degrades ulvan polysaccharides in the endolytic manner, mainly producing ΔRha3S, consisting of an unsaturated 4-deoxy-l-threo-hex-4-enopyranosiduronic acid and a 3-O-sulfated α-l-rhamnose. The structure of Uly1 was resolved at a 2.10-A resolution. Uly1 adopts a seven-bladed β-propeller architecture. Structural and site-directed mutagenesis analyses indicate that four highly conserved residues, H128, H149, Y223, and R239, are essential for catalysis. H128 functions as both the catalytic acid and base, H149 and R239 function as the neutralizers, and Y223 plays a supporting role in catalysis. Structural comparison and sequence alignment suggest that Uly1 and many other PL24 enzymes may directly bind the substrate near the catalytic residues for catalysis, different from the PL24 ulvan Lyase LOR_107, which adopts a two-stage substrate binding process. This study provides new insights into ulvan Lyases and ulvan degradation. IMPORTANCE Ulvan is a major cell wall component of green algae of the genus Ulva. Many marine heterotrophic bacteria can produce extracellular ulvan Lyases to degrade ulvan for a carbon nutrient. In addition, ulvan has a range of physiological bioactivities based on its specific chemical structure. Ulvan Lyase thus plays an important role in marine carbon cycling and has great potential in biotechnological applications. However, only a small number of ulvan Lyases have been characterized over the past 10 years. Here, based on biochemical and structural analyses, a new ulvan Lyase of polysaccharide Lyase family 24 is characterized, and its substrate recognition and catalytic mechanisms are revealed. Moreover, a new substrate binding process adopted by PL24 ulvan Lyases is proposed. This study offers a better understanding of bacterial ulvan Lyases and is helpful for studying the application potentials of ulvan Lyases.
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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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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, Xiying Zhang, Yuzhong Zhang, Sheng Dong, 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.
Sheng Dong - One of the best experts on this subject based on the ideXlab platform.
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structural basis for the exolytic activity of polysaccharide Lyase family 6 alginate Lyase bcalypl6 from human gut microbe bacteroides clarus
Biochemical and Biophysical Research Communications, 2021Co-Authors: Bing Wang, Sheng DongAbstract:Abstract Alginate is the structural polysaccharide of the cell wall of brown algae, which is an important carbon source for marine life. The depolymerization of alginate is dependent on alginate Lyases. Recent studies showed that the alginate utilization ability had been obtained by human gut microbes. In contrast to the great number of studies on alginate Lyases from marine/soil organisms, studies on alginate Lyases from gut microbes are still limited. Here, the structure of a polysaccharide Lyase family 6 (PL6) alginate Lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography, which represents the cluster of two-domain PL6 alginate Lyases from Bacteroidetes. Similar with the two-domain alginate Lyase AlyGC originated from marine bacterium, both the N terminal domain (NTD) and C terminal domain (CTD) of BcAlyPL6 show right-handed parallel β-helix fold. However, unlike AlyGC, which forms a homodimer, BcAlyPL6 functions as a monomer. Biochemical analysis indicates that the substrate binding affinity is mainly contributed by the NTD while the CTD of BcAlyPL6 is involved in the formation of −1 subsite, which is essential for substrate turnover rate. Furthermore, CTD is involved in shaping a closed catalytic pocket, and deletion of it leads to increased activity towards highly polymerized substrate. Structure comparison of PL6 family alginate Lyases implies that the linkers of two-domain alginate Lyases might have evolutionary relationship with the N/C terminal extension of single-domain Lyases.
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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, Xiying Zhang, Yuzhong Zhang, Sheng Dong, 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.
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cultivable alginate Lyase excreting bacteria associated with the arctic brown alga laminaria
Marine Drugs, 2012Co-Authors: Sheng Dong, Xiying Zhang, Xiaoyan Song, Xiulan Chen, Jie Yang, Yuzhong ZhangAbstract:Although some alginate Lyases have been isolated from marine bacteria, alginate Lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65), Psychromonas (10/65) and Polaribacter (8/65) were the predominant groups. Moreover, 21 alginate Lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate Lyase-excreting strains belong to five genera. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate Lyases. The optimal temperatures for the growth and algiante Lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate Lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate Lyase activity around 200 U/mL. These results suggest that these algiante Lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate Lyases.
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purification and characterization of a bifunctional alginate Lyase from pseudoalteromonas sp sm0524
Marine Drugs, 2011Co-Authors: Sheng Dong, Xiulan Chen, Jie Song, Binbin Xie, Yuzhong ZhangAbstract:Abstract: An alginate Lyase-producing bacterial strain, Pseudoalteromonas sp. SM0524, was screened from marine rotten kelp. In an optimized condition, the production of alginate Lyase from Pseudoalteromonas sp. SM0524 reached 62.6 U/mL, suggesting that strain SM0524 is a good producer of alginate Lyases. The bifunctional alginate Lyase aly-SJ02 secreted by strain SM0524 was purified. Aly-SJ02 had an apparent molecular mass of 32 kDa. The optimal temperature and pH of aly-SJ02 toward sodium alginate was 50 °C and 8.5, respectively. The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C . Aly-SJ02 was most stable at pH 8.0. N-terminal sequence analysis suggested that aly-SJ02 may be an alginate Lyase of polysaccharide Lyase family 18. Aly-SJ02 showed activities toward both polyG (α- L -guluronic acid) and polyM (β- D -mannuronic acid), indicating that it is a bifunctional alginate Lyase. Aly-SJ02 had lower
Kousaku Murata - One of the best experts on this subject based on the ideXlab platform.
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a novel structural fold in polysaccharide Lyases bacillus subtilis family 11 rhamnogalacturonan Lyase yesw with an eight bladed β propeller
Journal of Biological Chemistry, 2007Co-Authors: Akihito Ochiai, Wataru Hashimoto, Bunzo Mikami, Takafumi Itoh, Yukie Maruyama, Akiko Kawamata, Kousaku MurataAbstract:Abstract Rhamnogalacturonan (RG) Lyase produced by plant pathogenic and saprophytic microbes plays an important role in degrading plant cell walls. An extracellular RG Lyase YesW from saprophytic Bacillus subtilis is a member of polysaccharide Lyase family 11 and cleaves glycoside bonds in polygalacturonan as well as RG type-I through a β-elimination reaction. Crystal structures of YesW and its complex with galacturonan disaccharide, a reaction product analogue, were determined at 1.4 and 2.5A resolutions with final R-factors of 16.4% and 16.6%, respectively. The enzyme is composed of an eight-bladed β-propeller with a deep cleft in the center as a basic scaffold, and its structural fold has not been seen in polysaccharide Lyases analyzed thus far. Structural analysis of the disaccharide-bound YesW and a site-directed mutagenesis study suggested that Arg-452 and Lys-535 stabilize the carboxyl group of the acidic polysaccharide molecule and Tyr-595 makes a stack interaction with the sugar pyranose ring. In addition to amino acid residues binding to the disaccharide, one calcium ion, which is coordinated by Asp-401, Glu-422, His-363, and His-399, may mediate the enzyme activity. This is, to our knowledge, the first report of a new structural category with a β-propeller fold in polysaccharide Lyases and provides structural insights into substrate binding by RG Lyase.
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crystallization and preliminary x ray analysis of an exotype alginate Lyase atu3025 from agrobacterium tumefaciens strain c58 a member of polysaccharide Lyase family 15
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2006Co-Authors: Akihito Ochiai, Wataru Hashimoto, Masayuki Yamasaki, Bunzo Mikami, Kousaku MurataAbstract:Almost all alginate Lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate Lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate Lyase classified into polysaccharide Lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P21 and diffracted to 2.8 A resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 A, β = 91.5°.
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Crystallization and preliminary X-ray analysis of the rhamnogalacturonan Lyase YesW from Bacillus subtilis strain 168, a member of polysaccharide Lyase family 11.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Akihito Ochiai, Masayuki Yamasaki, Wataru Hashimoto, Bunzo Mikami, Takafumi Itoh, Kousaku MurataAbstract:Rhamnogalacturonan Lyases degrade rhamnogalacturonan I, a major component of pectin, through a β-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan Lyase classified into polysaccharide Lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P21 and diffract to 2.40 A resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 A, β = 94.9°. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan Lyase.
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Molecular identification of Sphingomonas sp. A1 alginate Lyase (A1-IV') as a member of novel polysaccharide Lyase family 15 and implications in alginate Lyase evolution.
Journal of Bioscience and Bioengineering, 2005Co-Authors: Wataru Hashimoto, Osamu Miyake, Akihito Ochiai, Kousaku MurataAbstract:Abstract Sphingomonas sp. A1 (strain A1) produces three endotypes (A1-I [65 kDa], A1-II [25 kDa], and A1-III [40 kDa]) and an exotype (A1-IV [86 kDa]) alginate Lyases in cytoplasm. These four enzymes cooperatively depolymerize alginate into constituent monosaccharides. In addition to the genes for these Lyases, novel genes encoding hypothetical proteins homologous with A1-IV were found in the genomes of many bacteria including strain A1. One such protein, A1-IV′ (90 kDa) of strain A1, was overexpressed in Escherichia coli cells, purified, and characterized. A1-IV′ catalyzed the cleavage of glycosidic bonds in alginate through a β-elimination reaction and released unsaturated di- and trisaccharides as main products, thus indicating that the enzyme is an endotype alginate Lyase. A1-IV′, which differed from A1-IV in some enzymatic properties, was not expressed in strain A1, suggesting that A1-IV′ has no significant role in alginate metabolism. A1-IV′ and other A1-IV homologs facilitate the creation of novel polysaccharide Lyase family 15 based on their primary structures, implying the evolution route of alginate Lyases in family PL-15.
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structure and function of a hypothetical pseudomonas aeruginosa protein pa1167 classified into family pl 7 a novel alginate Lyase with a β sandwich fold
Journal of Biological Chemistry, 2004Co-Authors: Masayuki Yamasaki, Kousaku Murata, Satoko Moriwaki, Osamu Miyake, Wataru Hashimoto, Bunzo MikamiAbstract:Abstract Structural and functional analyses of alginate Lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. Most alginate Lyases are classified into polysaccharide Lyase (PL) family-5 and -7 based on their primary structures. Family PL-7 enzymes are still poorly characterized especially in structural properties. Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate Lyase A1-II was found to be present in the P. aeruginosa genome. PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate Lyase catalyzing a β-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 °C. The specific activity of PA1167, however, was much weaker than that of the known alginate Lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa. In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. The first crystal structure of the family PL-7 alginate Lyase was determined at 2.0 A resolution. PA1167 was found to form a glove-like β-sandwich composed of 15 β-strands and 3 α-helices. The structural difference between the β-sandwich PA1167 of family PL-7 and α/α-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics. Crystal structures of polysaccharide Lyases determined so far indicate that they can be assigned to three folding groups having parallel β-helix, α/α-barrel, and α/α-barrel + antiparallel β-sheet structures as basic frames. PA1167 is the fourth novel folding structure found among polysaccharide Lyases.
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mechanistic insights into substrate recognition and catalysis of a new ulvan Lyase of the polysaccharide Lyase family 24
Applied and Environmental Microbiology, 2021Co-Authors: Fang Dong, Xiying Zhang, Yuzhong Zhang, Peng Wang, Haiyan Cao, Xiao-hui Sun, Andrew Mcminn, Xiulan ChenAbstract:Ulvan is an important marine polysaccharide. Bacterial ulvan Lyases play important roles in ulvan degradation and marine carbon cycling. Until now, only a small number of ulvan Lyases have been characterized. Here, a new ulvan Lyase, Uly1, belonging to polysaccharide Lyase family 24 (PL24) from the marine bacterium Catenovulum maritimum, is characterized. The optimal temperature and pH for Uly1 to degrade ulvan are 40°C and pH 9.0, respectively. Uly1 degrades ulvan polysaccharides in the endolytic manner, mainly producing ΔRha3S, consisting of an unsaturated 4-deoxy-l-threo-hex-4-enopyranosiduronic acid and a 3-O-sulfated α-l-rhamnose. The structure of Uly1 was resolved at a 2.10-A resolution. Uly1 adopts a seven-bladed β-propeller architecture. Structural and site-directed mutagenesis analyses indicate that four highly conserved residues, H128, H149, Y223, and R239, are essential for catalysis. H128 functions as both the catalytic acid and base, H149 and R239 function as the neutralizers, and Y223 plays a supporting role in catalysis. Structural comparison and sequence alignment suggest that Uly1 and many other PL24 enzymes may directly bind the substrate near the catalytic residues for catalysis, different from the PL24 ulvan Lyase LOR_107, which adopts a two-stage substrate binding process. This study provides new insights into ulvan Lyases and ulvan degradation. IMPORTANCE Ulvan is a major cell wall component of green algae of the genus Ulva. Many marine heterotrophic bacteria can produce extracellular ulvan Lyases to degrade ulvan for a carbon nutrient. In addition, ulvan has a range of physiological bioactivities based on its specific chemical structure. Ulvan Lyase thus plays an important role in marine carbon cycling and has great potential in biotechnological applications. However, only a small number of ulvan Lyases have been characterized over the past 10 years. Here, based on biochemical and structural analyses, a new ulvan Lyase of polysaccharide Lyase family 24 is characterized, and its substrate recognition and catalytic mechanisms are revealed. Moreover, a new substrate binding process adopted by PL24 ulvan Lyases is proposed. This study offers a better understanding of bacterial ulvan Lyases and is helpful for studying the application potentials of ulvan Lyases.
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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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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.
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Three Alginate Lyases from Marine Bacterium Pseudomonas fluorescens HZJ216: Purification and Characterization
Applied Biochemistry and Biotechnology, 2010Co-Authors: Xiaolu Jiang, Peng Wang, Huashi Guan, Hong GuoAbstract:Three alginate Lyases (A, B, and C) from an alginate-degrading marine bacterium strain HZJ216 isolated from brown seaweed in the Yellow Sea of China and identified preliminarily as Pseudomonas fluorescens are purified, and their biochemical properties are described. Molecular masses of the three enzymes are determined by SDS-PAGE to be 60.25, 36, and 23 kDa with isoelectric points of 4, 4.36, and 4.59, respectively. Investigations of these enzymes at different pH and temperatures show that they are most active at pH 7.0 and 35 °C. Alginate Lyases A and B are stable in the pH range of 5.0–9.0, while alginate Lyase C is stable in the pH range of 5.0–7.0. Among the metal ions tested, additions of Na+, K+, and Mg2+ ions can enhance the enzyme activities while Fe2+, Fe3+, Ba2+, and Zn2+ ions show inhibitory effects. The substrate specificity results demonstrate that alginate Lyase C has the specificity for G block while alginate Lyases A and B have the activities for both M and G blocks. It is the first report about extracellular alginate Lyases with high alginate-degrading activity from P. fluorescens.