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Kousaku Murata - One of the best experts on this subject based on the ideXlab platform.
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Alternative substrate-bound conformation of bacterial solute-binding protein involved in the import of mammalian host glycosaminoglycans
Scientific Reports, 2017Co-Authors: Sayoko Oiki, Kousaku Murata, Bunzo Mikami, Reiko Kamochi, Wataru HashimotoAbstract:Glycosaminoglycans (GAGs), constituted by repeating uronate and amino sugar units, are major components of mammalian extracellular matrices. Some indigenous and pathogenic bacteria target GAGs for colonization to and/or infection of host mammalian cells. In Gram-negative pathogenic Streptobacillus moniliformis , the solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter incorporates unsaturated GAG disaccharides into the cytoplasm after depolymerization by Polysaccharide Lyase. Smon0123, composed of N and C domains, adopts either a substrate-free open or a substrate-bound closed form by approaching two domains at 47° in comparison with the open form. Here we show an alternative 39°-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N -acetyl-d-galactosamine residue (CΔ4S6S). In CΔ4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47°-closed conformations. Therefore, the two sulfate groups in CΔ4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with CΔ4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of CΔ4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that CΔ4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.
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Crystal structure of exotype alginate Lyase Atu3025 from Agrobacterium tumefaciens
Journal of Biological Chemistry, 2010Co-Authors: Akihito Ochiai, Bunzo Mikami, Wataru Hashimoto, Masayuki Yamasaki, Kousaku MurataAbstract:Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate Lyases through a β-elimination reaction. Almost all alginate Lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate Lyase Atu3025, a member of Polysaccharide Lyase family 15, acts on alginate Polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/ΔGGG) were determined at 2.10- and 2.99-Å resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an α/α-barrel + anti-parallel β-sheet as a basic scaffold, and its structural fold has not been seen in alginate Lyases analyzed thus far. The structural analysis of H531A/ΔGGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His311 and Tyr365 as the catalytic base and acid, respectively. Two structural determinants, i.e. a short α-helix in the central α/α-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.
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crystal structure of family 14 Polysaccharide Lyase with ph dependent modes of action
Journal of Biological Chemistry, 2009Co-Authors: Kohei Ogura, Wataru Hashimoto, Bunzo Mikami, Masayuki Yamasaki, Takashi Yamada, Kousaku MurataAbstract:The Chlorella virus enzyme vAL-1 (38 kDa), a member of Polysaccharide Lyase family 14, degrades the Chlorella cell wall by cleaving the glycoside bond of the glucuronate residue (GlcA) through a beta-elimination reaction. The enzyme consists of an N-terminal cell wall-attaching domain (11 kDa) and a C-terminal catalytic module (27 kDa). Here, we show the enzyme characteristics of vAL-1, especially its pH-dependent modes of action, and determine the structure of the catalytic module. vAL-1 also exhibited alginate Lyase activity at alkaline pH, and truncation of the N-terminal domain increased the Lyase activity by 50-fold at pH 7.0. The truncated form vAL-1(S) released di- to hexasaccharides from alginate at pH 7.0, whereas disaccharides were preferentially generated at pH 10.0. This indicates that vAL-1(S) shows two pH-dependent modes of action: endo- and exotypes. The x-ray crystal structure of vAL-1(S) at 1.2 A resolution showed two antiparallel beta-sheets with a deep cleft showing a beta-jelly roll fold. The structure of GlcA-bound vAL-1(S) at pH 7.0 and 10.0 was determined: GlcA was found to be bound outside and inside the cleft at pH 7.0 and 10.0, respectively. This suggests that the electric charges at the active site greatly influence the binding mode of substrates and regulate endo/exo activity. Site-directed mutagenesis demonstrated that vAL-1(S) has a specific amino acid arrangement distinct from other alginate Lyases crucial for catalysis. This is, to our knowledge, the first study in which the structure of a family 14 Polysaccharide Lyase with two different modes of action has been determined.
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structural determinants responsible for substrate recognition and mode of action in family 11 Polysaccharide Lyases
Journal of Biological Chemistry, 2009Co-Authors: Akihito Ochiai, Bunzo Mikami, Wataru Hashimoto, Takafumi Itoh, Kousaku MurataAbstract:A saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) Lyases, endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide Lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a β-elimination reaction. Here we show the structural determinants for substrate recognition and the mode of action in Polysaccharide Lyase family 11 Lyases. The crystal structures of YesW in complex with rhamnose and ligand-free YesX were determined at 1.32 and 1.65 A resolution, respectively. The YesW amino acid residues such as Asn152, Asp172, Asn532, Gly533, Thr534, and Tyr595 in the active cleft bind to rhamnose molecules through hydrogen bonds and van der Waals contacts. Other rhamnose molecules are accommodated at the noncatalytic domain far from the active cleft, revealing that the domain possibly functions as a novel carbohydrate-binding module. A structural comparison between YesW and YesX indicates that a specific loop in YesX for recognizing the terminal saccharide molecule sterically inhibits penetration of the polymer over the active cleft. The loop-deficient YesX mutant exhibits YesW-like endotype activity, demonstrating that molecular conversion regarding the mode of action is achieved by the addition/removal of the loop for recognizing the terminal saccharide. This is the first report on a structural insight into RG-I recognition and molecular conversion of exotype to endotype in Polysaccharide Lyases.
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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.
Wataru Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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Alternative substrate-bound conformation of bacterial solute-binding protein involved in the import of mammalian host glycosaminoglycans
Scientific Reports, 2017Co-Authors: Sayoko Oiki, Kousaku Murata, Bunzo Mikami, Reiko Kamochi, Wataru HashimotoAbstract:Glycosaminoglycans (GAGs), constituted by repeating uronate and amino sugar units, are major components of mammalian extracellular matrices. Some indigenous and pathogenic bacteria target GAGs for colonization to and/or infection of host mammalian cells. In Gram-negative pathogenic Streptobacillus moniliformis , the solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter incorporates unsaturated GAG disaccharides into the cytoplasm after depolymerization by Polysaccharide Lyase. Smon0123, composed of N and C domains, adopts either a substrate-free open or a substrate-bound closed form by approaching two domains at 47° in comparison with the open form. Here we show an alternative 39°-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N -acetyl-d-galactosamine residue (CΔ4S6S). In CΔ4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47°-closed conformations. Therefore, the two sulfate groups in CΔ4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with CΔ4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of CΔ4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that CΔ4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.
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Crystal structure of exotype alginate Lyase Atu3025 from Agrobacterium tumefaciens
Journal of Biological Chemistry, 2010Co-Authors: Akihito Ochiai, Bunzo Mikami, Wataru Hashimoto, Masayuki Yamasaki, Kousaku MurataAbstract:Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate Lyases through a β-elimination reaction. Almost all alginate Lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate Lyase Atu3025, a member of Polysaccharide Lyase family 15, acts on alginate Polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/ΔGGG) were determined at 2.10- and 2.99-Å resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an α/α-barrel + anti-parallel β-sheet as a basic scaffold, and its structural fold has not been seen in alginate Lyases analyzed thus far. The structural analysis of H531A/ΔGGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His311 and Tyr365 as the catalytic base and acid, respectively. Two structural determinants, i.e. a short α-helix in the central α/α-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.
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crystal structure of family 14 Polysaccharide Lyase with ph dependent modes of action
Journal of Biological Chemistry, 2009Co-Authors: Kohei Ogura, Wataru Hashimoto, Bunzo Mikami, Masayuki Yamasaki, Takashi Yamada, Kousaku MurataAbstract:The Chlorella virus enzyme vAL-1 (38 kDa), a member of Polysaccharide Lyase family 14, degrades the Chlorella cell wall by cleaving the glycoside bond of the glucuronate residue (GlcA) through a beta-elimination reaction. The enzyme consists of an N-terminal cell wall-attaching domain (11 kDa) and a C-terminal catalytic module (27 kDa). Here, we show the enzyme characteristics of vAL-1, especially its pH-dependent modes of action, and determine the structure of the catalytic module. vAL-1 also exhibited alginate Lyase activity at alkaline pH, and truncation of the N-terminal domain increased the Lyase activity by 50-fold at pH 7.0. The truncated form vAL-1(S) released di- to hexasaccharides from alginate at pH 7.0, whereas disaccharides were preferentially generated at pH 10.0. This indicates that vAL-1(S) shows two pH-dependent modes of action: endo- and exotypes. The x-ray crystal structure of vAL-1(S) at 1.2 A resolution showed two antiparallel beta-sheets with a deep cleft showing a beta-jelly roll fold. The structure of GlcA-bound vAL-1(S) at pH 7.0 and 10.0 was determined: GlcA was found to be bound outside and inside the cleft at pH 7.0 and 10.0, respectively. This suggests that the electric charges at the active site greatly influence the binding mode of substrates and regulate endo/exo activity. Site-directed mutagenesis demonstrated that vAL-1(S) has a specific amino acid arrangement distinct from other alginate Lyases crucial for catalysis. This is, to our knowledge, the first study in which the structure of a family 14 Polysaccharide Lyase with two different modes of action has been determined.
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structural determinants responsible for substrate recognition and mode of action in family 11 Polysaccharide Lyases
Journal of Biological Chemistry, 2009Co-Authors: Akihito Ochiai, Bunzo Mikami, Wataru Hashimoto, Takafumi Itoh, Kousaku MurataAbstract:A saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) Lyases, endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide Lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a β-elimination reaction. Here we show the structural determinants for substrate recognition and the mode of action in Polysaccharide Lyase family 11 Lyases. The crystal structures of YesW in complex with rhamnose and ligand-free YesX were determined at 1.32 and 1.65 A resolution, respectively. The YesW amino acid residues such as Asn152, Asp172, Asn532, Gly533, Thr534, and Tyr595 in the active cleft bind to rhamnose molecules through hydrogen bonds and van der Waals contacts. Other rhamnose molecules are accommodated at the noncatalytic domain far from the active cleft, revealing that the domain possibly functions as a novel carbohydrate-binding module. A structural comparison between YesW and YesX indicates that a specific loop in YesX for recognizing the terminal saccharide molecule sterically inhibits penetration of the polymer over the active cleft. The loop-deficient YesX mutant exhibits YesW-like endotype activity, demonstrating that molecular conversion regarding the mode of action is achieved by the addition/removal of the loop for recognizing the terminal saccharide. This is the first report on a structural insight into RG-I recognition and molecular conversion of exotype to endotype in Polysaccharide Lyases.
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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.
Takao Ojima - One of the best experts on this subject based on the ideXlab platform.
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structure and polymannuronate specificity of a eukaryotic member of Polysaccharide Lyase family 14
Journal of Biological Chemistry, 2017Co-Authors: Takuya Miyakawa, Akira Inoue, Ryuji Nishiyama, Akira Nakamura, Atsuko Asano, Yoriko Sawano, Takao Ojima, Masaru TanokuraAbstract:: Alginate is an abundant algal Polysaccharide, composed of β-d-mannuronate and its C5 epimer α-l-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate Lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the Polysaccharide Lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a β-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 A resolution and the putative substrate-binding model show that the enzyme adopts a β-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the β-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115-Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate Lyases.
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characterization of an alginate Lyase flalya from flavobacterium sp strain umi 01 and its expression in escherichia coli
Marine Drugs, 2014Co-Authors: Akira Inoue, Ryuji Nishiyama, Kohei Takadono, Kenji Tajima, Takanori Kobayashi, Takao OjimaAbstract:Abstract: A major alginate Lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) Lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N -terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C -terminal region of 260 residues was regarded as the Polysaccharide-Lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I— Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of
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characterization of an alginate Lyase flalya from flavobacterium sp strain umi 01 and its expression in escherichia coli
Marine Drugs, 2014Co-Authors: Akira Inoue, Ryuji Nishiyama, Kohei Takadono, Kenji Tajima, Takanori Kobayashi, Takao OjimaAbstract:A major alginate Lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) Lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the Polysaccharide-Lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.
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, Xiaohui 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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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.
Akira Inoue - One of the best experts on this subject based on the ideXlab platform.
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Characterization of PL-7 Family Alginate Lyases From Marine Organisms and Their Applications.
Marine Enzymes and Specialized Metabolism - Part B, 2018Co-Authors: Akira InoueAbstract:Alginate, an anionic heteroPolysaccharide extracted from natural brown algae, has useful properties for the food, chemical, medical, and agricultural industries. Degradation of alginate by alginate Lyase is a key process to produce unsaturated oligoalginate and unsaturated monosaccharide 4-deoxy-l-erythro-5-hexoseulose uronic acid. Alginate Lyases belonging to the Polysaccharide Lyase family 7 have been found in, and isolated from, organisms thriving in various environments. Furthermore, research on their function and structure has also progressed well. Here, the preparation of native and recombinant PL-7 alginate Lyases and the methods for evaluation of enzymatic activity are summarized. Examples of PL-7 alginate Lyase applications are also described.
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structure and polymannuronate specificity of a eukaryotic member of Polysaccharide Lyase family 14
Journal of Biological Chemistry, 2017Co-Authors: Takuya Miyakawa, Akira Inoue, Ryuji Nishiyama, Akira Nakamura, Atsuko Asano, Yoriko Sawano, Takao Ojima, Masaru TanokuraAbstract:: Alginate is an abundant algal Polysaccharide, composed of β-d-mannuronate and its C5 epimer α-l-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate Lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the Polysaccharide Lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a β-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 A resolution and the putative substrate-binding model show that the enzyme adopts a β-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the β-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115-Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate Lyases.
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characterization of an alginate Lyase flalya from flavobacterium sp strain umi 01 and its expression in escherichia coli
Marine Drugs, 2014Co-Authors: Akira Inoue, Ryuji Nishiyama, Kohei Takadono, Kenji Tajima, Takanori Kobayashi, Takao OjimaAbstract:Abstract: A major alginate Lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) Lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N -terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C -terminal region of 260 residues was regarded as the Polysaccharide-Lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I— Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of
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characterization of an alginate Lyase flalya from flavobacterium sp strain umi 01 and its expression in escherichia coli
Marine Drugs, 2014Co-Authors: Akira Inoue, Ryuji Nishiyama, Kohei Takadono, Kenji Tajima, Takanori Kobayashi, Takao OjimaAbstract:A major alginate Lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) Lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the Polysaccharide-Lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.