scholarly journals Investigation of Action Pattern of a Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-Endosulfatase

2020 ◽  
Author(s):  
Wenshuang Wang ◽  
Cédric Przybylski ◽  
Xiaojuan Cai ◽  
Chrystel Lopin-Bon ◽  
Runmiao Jiao ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Marine Bacterium ◽  
Catalytic Mechanism ◽  
Dermatan Sulfate ◽  
Structural Characteristics ◽  
Action Pattern ◽  
Substrate Specificities ◽  
Substrate Size ◽  
Function Relationship ◽  
Relationship Of

Recently, a novel CS/DS 4-O-endosulfatase was identified from a marine bacterium and its catalytic mechanism was investigated further (Wang, W., et.al (2015) J. Biol. Chem. 290, 7823-7832; Wang, S., et.al (2019) Front. Microbiol. 10:1309). In the study herein, we provide new insight about the structural characteristics of substrate which determine the activity of this enzyme. The substrate specificities of the 4-O-endosulfatase were probed by using libraries of structure-defined CS/DS oligosacccharides issued from synthetic and enzymatic sources. We found that this 4-O-endosulfatase effectively remove the 4-O-sulfate of disaccharide sequences GlcUAβ1-3GalNAc(4S) or GlcUAβ1-3GalNAc(4S,6S) in all tested hexasaccharides. The sulfated GalNac residue is resistant to the enzyme when adjacent uronic residues are sulfated as shown by the lack of enzymatic desulfation of GlcUAβ1-3GalNAc(4S) connected to a disaccharide GlcUA(2S)β1-3GalNAc(6S) in an octasaccharide. The 3-O-sulfation of GlcUA was also shown to hinder the action of this enzyme. The 4-O-endosulfatase exhibited an oriented action from the reducing to the non-reducing whatever the saturation or not of the non-reducing end. Finally, the activity of the 4-O-endosulfatase decreases with the increase of substrate size. With the deeper understanding of this novel 4-O-endosulfatase, such chondroitin sulfate (CS)/dermatan sulfate (DS) sulfatase is a useful tool for exploring the structure-function relationship of CS/DS.

Sequencing of chondroitin sulfate oligosaccharides using a novel exolyase from a marine bacterium that degrades hyaluronan and chondroitin sulfate/dermatan sulfate

2017 ◽  
Vol 474 (22) ◽  
pp. 3831-3848 ◽  
Author(s):  
Wenshuang Wang ◽  
Xiaojuan Cai ◽  
Naihan Han ◽  
Wenjun Han ◽  
Kazuyuki Sugahara ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Marine Bacterium ◽  
Dermatan Sulfate ◽  
Specific Activity ◽  
Structural Complexity ◽  
Complex Structures ◽  
Biological Functions ◽  
Chemical Structures ◽  
Lyase Activity ◽  
Sugar Chains

Glycosaminoglycans (GAGs) are a family of chemically heterogeneous polysaccharides that play important roles in physiological and pathological processes. Owing to the structural complexity of GAGs, their sophisticated chemical structures and biological functions have not been extensively studied. Lyases that cleave GAGs are important tools for structural analysis. Although various GAG lyases have been identified, exolytic lyases with unique enzymatic property are urgently needed for GAG sequencing. In the present study, a putative exolytic GAG lyase from a marine bacterium was recombinantly expressed and characterized in detail. Since it showed exolytic lyase activity toward hyaluronan (HA), chondroitin sulfate (CS), and dermatan sulfate (DS), it was designated as HCDLase. This novel exolyase exhibited the highest activity in Tris–HCl buffer (pH 7.0) at 30°C. Especially, it showed a specific activity that released 2-aminobenzamide (2-AB)-labeled disaccharides from the reducing end of 2-AB-labeled CS oligosaccharides, which suggest that HCDLase is not only a novel exolytic lyase that can split disaccharide residues from the reducing termini of sugar chains but also a useful tool for the sequencing of CS chains. Notably, HCDLase could not digest 2-AB-labeled oligosaccharides from HA, DS, or unsulfated chondroitin, which indicated that sulfates and bond types affect the catalytic activity of HCDLase. Finally, this enzyme combined with CSase ABC was successfully applied for the sequencing of several CS hexa- and octasaccharides with complex structures. The identification of HCDLase provides a useful tool for CS-related research and applications.

scholarly journals Exploring the binding properties and structural stability of an opsin in the chytrid Spizellomyces punctatus using comparative and molecular modeling

2017 ◽  
Author(s):  
Steven R Ahrendt ◽  
Edgar Mauricio Medina ◽  
Chia-en A Chang ◽  
Jason E Stajich
Keyword(s):  
Molecular Modeling ◽  
Structural Characteristics ◽  
Sequence Similarity ◽  
Ligand Docking ◽  
Binding Properties ◽  
Function Relationship ◽  
Relationship Of ◽  
Future Work

Background. Opsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores. Methods. We used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found in Spizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squid Todarodes pacificus. Results. Our results indicate that the Spizellomyces opsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis­-retinal isomer. Together, these results support further the homology of Spizellomyces opsins to animal type 2 rhodopsins. Discussion. This represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi.

scholarly journals Exploring the binding properties and structural stability of an opsin in the chytridSpizellomyces punctatususing comparative and molecular modeling

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3206 ◽  
Author(s):  
Steven R. Ahrendt ◽  
Edgar Mauricio Medina ◽  
Chia-en A. Chang ◽  
Jason E. Stajich
Keyword(s):  
Molecular Modeling ◽  
Structural Characteristics ◽  
Sequence Similarity ◽  
Ligand Docking ◽  
Binding Properties ◽  
Function Relationship ◽  
Relationship Of ◽  
Future Work

BackgroundOpsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores.MethodsWe used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found inSpizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if the sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squidTodarodes pacificus.ResultsOur results indicate that theSpizellomycesopsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis-retinal isomer. Together, these results support further the homology ofSpizellomycesopsins to animal type 2 rhodopsins.DiscussionThis represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi.

scholarly journals Exploring the binding properties and structural stability of an opsin in the chytrid Spizellomyces punctatus using comparative and molecular modeling

2016 ◽  
Author(s):  
Steven R Ahrendt ◽  
Edgar Mauricio Medina ◽  
Chia-en A Chang ◽  
Jason E Stajich
Keyword(s):  
Molecular Modeling ◽  
Structural Characteristics ◽  
Sequence Similarity ◽  
Ligand Docking ◽  
Binding Properties ◽  
Function Relationship ◽  
Relationship Of ◽  
Future Work

Background. Opsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores. Methods. We used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found in Spizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squid Todarodes pacificus. Results. Our results indicate that the Spizellomyces opsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis­-retinal isomer. Together, these results support further the homology of Spizellomyces opsins to animal type 2 rhodopsins. Discussion. This represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi.

The chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from marine bacterium Vibrio sp FC509 is a dimeric species: Biophysical characterization of an endosulfatase

Biochimie ◽  
2016 ◽  
Vol 131 ◽  
pp. 85-95 ◽  
Author(s):  
José L. Neira ◽  
Encarnación Medina-Carmona ◽  
José G. Hernández-Cifre ◽  
Laia Montoliu-Gaya ◽  
Ana Cámara-Artigás ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Marine Bacterium ◽  
Dermatan Sulfate ◽  
Biophysical Characterization

Characterization of the pits in parenchyma cells of the moso bamboo [Phyllostachys edulis (Carr.) J. Houz.] culm

Holzforschung ◽  
2019 ◽  
Vol 73 (7) ◽  
pp. 629-636 ◽  
Author(s):  
Caiping Lian ◽  
Rong Liu ◽  
Cheng Xiufang ◽  
Shuqing Zhang ◽  
Junji Luo ◽  
...  
Keyword(s):  
Structural Characteristics ◽  
Parenchyma Cell ◽  
Environmental Scanning Electron Microscopy ◽  
Environmental Scanning ◽  
Bordered Pits ◽  
Parenchyma Cells ◽  
Vascular Parenchyma ◽  
Function Relationship ◽  
Relationship Of ◽  
First Time

Abstract The pits on parenchyma cell walls facilitate transfer of liquids between adjacent cells in the bamboo. To better understand the structure-function relationship of the pits, the structural characteristics of the pits in bamboo parenchyma cells need to be investigated. In this study, the pit structures were studied by field-emission environmental scanning electron microscopy (SEM). The samples included the native structure and the replica structure via resin castings. The results showed that the parenchyma cells possessed various shapes and the pits were diverse. Parenchyma cells exposed both simple and bordered pits. Pitting between vascular parenchyma cells (VPCs) was similar to that of the metaxylem vessel. In particular, a branched pit structure was found for the first time in the parenchyma cell.

scholarly journals Cloning and Characterization of a Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-Endosulfatase from a Marine Bacterium

2015 ◽  
Vol 290 (12) ◽  
pp. 7823-7832 ◽  
Author(s):  
Wenshuang Wang ◽  
Wenjun Han ◽  
Xingya Cai ◽  
Xiaoyu Zheng ◽  
Kazuyuki Sugahara ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Marine Bacterium ◽  
Dermatan Sulfate

scholarly journals Structural insights into the catalytic mechanism of lovastatin hydrolase

2019 ◽  
Vol 295 (4) ◽  
pp. 1047-1055
Author(s):  
Yajing Liang ◽  
Xuefeng Lu
Keyword(s):  
Structure Function ◽  
Catalytic Mechanism ◽  
Catalytic Triad ◽  
Enzyme Engineering ◽  
Rapid Screening ◽  
Nucleophilic Attack ◽  
Specific Substrate ◽  
Structure Function Relationship ◽  
Function Relationship ◽  
Relationship Of

The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure–function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)–Tyr170 (general base)–Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2′,2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure–function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.

scholarly journals Exploring the binding properties and structural stability of an opsin in the chytrid Spizellomyces punctatus using comparative and molecular modeling

2017 ◽  
Author(s):  
Steven R Ahrendt ◽  
Edgar Mauricio Medina ◽  
Chia-en A Chang ◽  
Jason E Stajich
Keyword(s):  
Molecular Modeling ◽  
Structural Characteristics ◽  
Sequence Similarity ◽  
Ligand Docking ◽  
Binding Properties ◽  
Function Relationship ◽  
Relationship Of ◽  
Future Work

Background. Opsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores. Methods. We used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found in Spizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squid Todarodes pacificus. Results. Our results indicate that the Spizellomyces opsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis­-retinal isomer. Together, these results support further the homology of Spizellomyces opsins to animal type 2 rhodopsins. Discussion. This represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi.

scholarly journals Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7716 ◽  
Author(s):  
Boxing Cheng ◽  
Fei Liu ◽  
Qiaosheng Guo ◽  
Yuxi Lu ◽  
Hongzhuan Shi ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Disulfide Bonds ◽  
Interaction Analysis ◽  
Structural Characteristics ◽  
Anticoagulant Activity ◽  
Thrombin Inhibitor ◽  
Sequencing Data ◽  
Function Relationship ◽  
Terminal Structure ◽  
Relationship Of

Transcriptome sequencing data (6.5 Gb) of the salivary glands of the haematophagous leech Hirudo nipponia was obtained by using the BGIseq-500 platform. After identification and analysis, one transcript (Unigene5370) was annotated to hirudin HV3 from Hirudo medicinalis with an e-value of 1e-29 and was named hirudin-HN. This transcript was a new thrombin inhibitor gene belonging to the proteinase inhibitor I14 (hirudin) family. Hirudin-HN, with a 270-bp cDNA, encodes an 89-aa protein containing a 20-aa signal peptide. The mature hirudin-HN protein contains the typical structural characteristics of hirudin, e.g., three conserved disulfide bonds and the PKP and DFxxIP motifs. Proteins (Hir and M-Hir) were obtained via prokaryotic expression, and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance (SPR) interaction analysis, respectively. The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity of Hir and M-Hir. The abundances of Hirudin-HN mRNA and protein were higher in the salivary glands of starving animals than in those of feeding or fed leeches. These results provided a foundation for further study on the structure-function relationship of hirudin-HN with thrombin.

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