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Isabel Correia - One of the best experts on this subject based on the ideXlab platform.
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Cloning, expression, purification, crystallization and preliminary crystallographic studies of UgdG, an UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009Co-Authors: Joana Rocha, Ana Teresa Granja, Isabel Correia, Arsenio M Fialho, Carlos FrazaoAbstract:Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 1.1.1.22) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 A resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein.
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Structural analysis of gellans produced by Sphingomonas elodea strains by electrospray tandem mass spectrometry
Carbohydrate Polymers, 2009Co-Authors: Virgínia M.f. Gonçalves, Leonilde M. Moreira, Isabel Correia, Arsenio M Fialho, Ana Reis, M. Rosário M. Domingues, José A. Lopes-da-silva, Manuel A. CoimbraAbstract:Abstract A commercial gellan sample (Gelrite) and a gellan-like polymer (JB3) obtained by exposure of the producing strain to chemical mutagenesis were subjected to partial acid hydrolysis and the resultant oligosaccharides were identified by Electrospray Mass Spectrometry (ESI-MS) and Tandem Mass Spectrometry (ESI-MS/MS and MS n ). In both gellans, the main fragments were in accordance with the tetrasaccharide repeating unit, d -Glucose (Glc)- d -Glucuronic acid (GlcA)- d -Glucose (Glc)- l -Rhamose (Rha), described for the wild-type gellan, showing the higher acid lability of the Rha-(1 → 3)-Glc linkage when compared to the (1 → 4) of the other residues. Under the experimental conditions used in the study, as expected, no acyl substituents were observed in the commercial gellan but in JB3 oligosaccharides a glyceryl moiety was identified, substituted in the 3-linked Glc residue. Furthermore, the analysis of the MS/MS and MS n spectra of both gellans allowed the identification of structural details, some of them not yet reported for these exopolysaccharides. The presence of oligosaccharides with single Glc and Rha residues substituent of the tetrasaccharide unit of gellan may represent novel side chains of the backbone unit that to our knowledge have never been reported previously for the gellan exopolysaccharide.
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Occurrence, production, and applications of gellan: current state and perspectives
Applied Microbiology and Biotechnology, 2008Co-Authors: Alexandre Madi Fialho, Leonilde M. Moreira, Ana Teresa Granja, Alma O. Popescu, Karen Hoffmann, Isabel CorreiaAbstract:Bacterial exopolysaccharides (EPS) are products of biotechnology that are of high interest due to their rheological properties. This is the case of sphingans, a group of structurally related EPS secreted by members of the genus Sphingomonas . Among these, gellan is a multifunctional gelling agent produced in high yields by the non-pathogenic strain Sphingomonas elodea ATCC 31461. In its native form, gellan is a linear anionic EPS based on a tetrasaccharide repeat unit composed of two molecules of d -glucose, one of l -rhamnose and one of d -glucuronic acid. The native gellan is partially esterified with acyl substituents (1 mol of glycerate and 0.5 mol of acetate) per repeat unit. Gellan has unique characteristics and has many applications, particularly in the food, pharmaceutical, and biomedical fields. This review summarizes current knowledge on the structure and properties of gellan and provides details about the biosynthesis of this exopolysaccharide. In addition, a highlight of the importance of gellan in industrial and medicinal applications is given.
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Biochemical characterization and phylogenetic analysis of UDP-glucose dehydrogenase from the gellan gum producer Sphingomonas elodea ATCC 31461
Applied Microbiology and Biotechnology, 2007Co-Authors: Ana Teresa Granja, Isabel Correia, Alma Popescu, Ana Rita Marques, Arsenio M FialhoAbstract:Sphingomonas elodea ATCC 31461 synthesizes in high yield the exopolysaccharide gellan, which is a water-soluble gelling agent with many applications. In this study, we describe the cloning and sequence analysis of the ugdG gene, encoding a UDP-glucose dehydrogenase (47.2 kDa; UDPG-DH; EC 1.1.1.22), required for the synthesis of the gellan gum precursor UDP-glucuronic acid. UgdG protein shows homology to members of the UDP-glucose/GDP-mannose dehydrogenase superfamily. The Neighbor-Joining method was used to determine phylogenetic relationships among prokaryotic and eukaryotic UDPG-DHs. UgdG from S. elodea and UDPG-DHs from Novosphingobium , Zymomonas , Agrobacterium , and Caulobacter species form a divergent phylogenetic group with a close evolutionary relationship with eukaryotic UDPG-DHs. The ugdG gene was recombinantly expressed in Escherichia coli with and N-terminal 6-His tag and purified for biochemical characterization. The enzyme has an optimum temperature and pH of 37°C and 8.7, respectively. The estimated apparent K _m values for UDP-glucose and NAD^+ were 0.87 and 0.4 mM, respectively. DNA sequencing of chromosomal regions adjacent to ugdG gene and sequence similarity studies suggests that this gene maps together with others presumably involved in the biosynthesis of S. elodea cell wall polysaccharides.
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Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: David Aragao, Isabel Correia, Arsenio M Fialho, A R Marques, Carlos F. Frazao, Francisco J. Enguita, Maria Arménia Carrondo, Edward P MitchellAbstract:The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P21 crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P21 has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, β = 105.2°. Model building and refinement are currently under way.
Arsenio M Fialho - One of the best experts on this subject based on the ideXlab platform.
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Rossmann-fold motifs can confer multiple functions to metabolic enzymes: RNA binding and ribonuclease activity of a UDP-glucose dehydrogenase.
Biochemical and biophysical research communications, 2012Co-Authors: Ana Barbas, Alma O. Popescu, Carlos Frazao, Cecília M. Arraiano, Arsenio M FialhoAbstract:Metabolic enzymes are usually characterized to have one specific function, and this is the case of UDP-glucose dehydrogenase that catalyzes the twofold NAD+-dependent oxidation of UDP-glucose into UDP-glucuronic acid. We have determined that this enzyme is also capable of participating in other cellular processes. Here, we report that the bacterial UDP-glucose dehydrogenase (UgdG) from Sphingomonas elodea ATCC 31461, which provides UDP-glucuronic acid for the synthesis of the exopolysaccharide gellan, is not only able to bind RNA but also acts as a ribonuclease. The ribonucleolytic activity occurs independently of the presence of NAD+ and the RNA binding site does not coincide with the NAD+ binding region. We have also performed the kinetics of interaction between UgdG and RNA. Moreover, computer analysis reveals that the N- and C-terminal domains of UgdG share structural features with ancient mitochondrial ribonucleases named MAR. MARs are present in lower eukaryotic microorganisms, have a Rossmannoid-fold and belong to the isochorismatase superfamily. This observation reinforces that the Rossmann structural motifs found in NAD+-dependent dehydrogenases can have a dual function working as a nucleotide cofactor binding domain and as a ribonuclease.
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Cloning, expression, purification, crystallization and preliminary crystallographic studies of UgdG, an UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009Co-Authors: Joana Rocha, Ana Teresa Granja, Isabel Correia, Arsenio M Fialho, Carlos FrazaoAbstract:Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 1.1.1.22) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 A resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein.
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Structural analysis of gellans produced by Sphingomonas elodea strains by electrospray tandem mass spectrometry
Carbohydrate Polymers, 2009Co-Authors: Virgínia M.f. Gonçalves, Leonilde M. Moreira, Isabel Correia, Arsenio M Fialho, Ana Reis, M. Rosário M. Domingues, José A. Lopes-da-silva, Manuel A. CoimbraAbstract:Abstract A commercial gellan sample (Gelrite) and a gellan-like polymer (JB3) obtained by exposure of the producing strain to chemical mutagenesis were subjected to partial acid hydrolysis and the resultant oligosaccharides were identified by Electrospray Mass Spectrometry (ESI-MS) and Tandem Mass Spectrometry (ESI-MS/MS and MS n ). In both gellans, the main fragments were in accordance with the tetrasaccharide repeating unit, d -Glucose (Glc)- d -Glucuronic acid (GlcA)- d -Glucose (Glc)- l -Rhamose (Rha), described for the wild-type gellan, showing the higher acid lability of the Rha-(1 → 3)-Glc linkage when compared to the (1 → 4) of the other residues. Under the experimental conditions used in the study, as expected, no acyl substituents were observed in the commercial gellan but in JB3 oligosaccharides a glyceryl moiety was identified, substituted in the 3-linked Glc residue. Furthermore, the analysis of the MS/MS and MS n spectra of both gellans allowed the identification of structural details, some of them not yet reported for these exopolysaccharides. The presence of oligosaccharides with single Glc and Rha residues substituent of the tetrasaccharide unit of gellan may represent novel side chains of the backbone unit that to our knowledge have never been reported previously for the gellan exopolysaccharide.
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Biochemical characterization and phylogenetic analysis of UDP-glucose dehydrogenase from the gellan gum producer Sphingomonas elodea ATCC 31461
Applied Microbiology and Biotechnology, 2007Co-Authors: Ana Teresa Granja, Isabel Correia, Alma Popescu, Ana Rita Marques, Arsenio M FialhoAbstract:Sphingomonas elodea ATCC 31461 synthesizes in high yield the exopolysaccharide gellan, which is a water-soluble gelling agent with many applications. In this study, we describe the cloning and sequence analysis of the ugdG gene, encoding a UDP-glucose dehydrogenase (47.2 kDa; UDPG-DH; EC 1.1.1.22), required for the synthesis of the gellan gum precursor UDP-glucuronic acid. UgdG protein shows homology to members of the UDP-glucose/GDP-mannose dehydrogenase superfamily. The Neighbor-Joining method was used to determine phylogenetic relationships among prokaryotic and eukaryotic UDPG-DHs. UgdG from S. elodea and UDPG-DHs from Novosphingobium , Zymomonas , Agrobacterium , and Caulobacter species form a divergent phylogenetic group with a close evolutionary relationship with eukaryotic UDPG-DHs. The ugdG gene was recombinantly expressed in Escherichia coli with and N-terminal 6-His tag and purified for biochemical characterization. The enzyme has an optimum temperature and pH of 37°C and 8.7, respectively. The estimated apparent K _m values for UDP-glucose and NAD^+ were 0.87 and 0.4 mM, respectively. DNA sequencing of chromosomal regions adjacent to ugdG gene and sequence similarity studies suggests that this gene maps together with others presumably involved in the biosynthesis of S. elodea cell wall polysaccharides.
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the complex of Sphingomonas elodea atcc 31461 glucose 1 phosphate uridylyltransferase with glucose 1 phosphate reveals a novel quaternary structure unique among nucleoside diphosphate sugar pyrophosphorylase members
Journal of Bacteriology, 2007Co-Authors: David Aragao, Arsenio M Fialho, A R Marques, Edward P Mitchell, Isabel Sacorreia, Carlos FrazaoAbstract:Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.
David Aragao - One of the best experts on this subject based on the ideXlab platform.
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the complex of Sphingomonas elodea atcc 31461 glucose 1 phosphate uridylyltransferase with glucose 1 phosphate reveals a novel quaternary structure unique among nucleoside diphosphate sugar pyrophosphorylase members
Journal of Bacteriology, 2007Co-Authors: David Aragao, Arsenio M Fialho, A R Marques, Edward P Mitchell, Isabel Sacorreia, Carlos FrazaoAbstract:Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.
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Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: David Aragao, Isabel Correia, Arsenio M Fialho, A R Marques, Carlos F. Frazao, Francisco J. Enguita, Maria Arménia Carrondo, Edward P MitchellAbstract:The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P21 crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P21 has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, β = 105.2°. Model building and refinement are currently under way.
Carlos Frazao - One of the best experts on this subject based on the ideXlab platform.
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Rossmann-fold motifs can confer multiple functions to metabolic enzymes: RNA binding and ribonuclease activity of a UDP-glucose dehydrogenase.
Biochemical and biophysical research communications, 2012Co-Authors: Ana Barbas, Alma O. Popescu, Carlos Frazao, Cecília M. Arraiano, Arsenio M FialhoAbstract:Metabolic enzymes are usually characterized to have one specific function, and this is the case of UDP-glucose dehydrogenase that catalyzes the twofold NAD+-dependent oxidation of UDP-glucose into UDP-glucuronic acid. We have determined that this enzyme is also capable of participating in other cellular processes. Here, we report that the bacterial UDP-glucose dehydrogenase (UgdG) from Sphingomonas elodea ATCC 31461, which provides UDP-glucuronic acid for the synthesis of the exopolysaccharide gellan, is not only able to bind RNA but also acts as a ribonuclease. The ribonucleolytic activity occurs independently of the presence of NAD+ and the RNA binding site does not coincide with the NAD+ binding region. We have also performed the kinetics of interaction between UgdG and RNA. Moreover, computer analysis reveals that the N- and C-terminal domains of UgdG share structural features with ancient mitochondrial ribonucleases named MAR. MARs are present in lower eukaryotic microorganisms, have a Rossmannoid-fold and belong to the isochorismatase superfamily. This observation reinforces that the Rossmann structural motifs found in NAD+-dependent dehydrogenases can have a dual function working as a nucleotide cofactor binding domain and as a ribonuclease.
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Cloning, expression, purification, crystallization and preliminary crystallographic studies of UgdG, an UDP-glucose dehydrogenase from Sphingomonas elodea ATCC 31461
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009Co-Authors: Joana Rocha, Ana Teresa Granja, Isabel Correia, Arsenio M Fialho, Carlos FrazaoAbstract:Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 1.1.1.22) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 A resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein.
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the complex of Sphingomonas elodea atcc 31461 glucose 1 phosphate uridylyltransferase with glucose 1 phosphate reveals a novel quaternary structure unique among nucleoside diphosphate sugar pyrophosphorylase members
Journal of Bacteriology, 2007Co-Authors: David Aragao, Arsenio M Fialho, A R Marques, Edward P Mitchell, Isabel Sacorreia, Carlos FrazaoAbstract:Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.
A R Marques - One of the best experts on this subject based on the ideXlab platform.
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the complex of Sphingomonas elodea atcc 31461 glucose 1 phosphate uridylyltransferase with glucose 1 phosphate reveals a novel quaternary structure unique among nucleoside diphosphate sugar pyrophosphorylase members
Journal of Bacteriology, 2007Co-Authors: David Aragao, Arsenio M Fialho, A R Marques, Edward P Mitchell, Isabel Sacorreia, Carlos FrazaoAbstract:Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.
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Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: David Aragao, Isabel Correia, Arsenio M Fialho, A R Marques, Carlos F. Frazao, Francisco J. Enguita, Maria Arménia Carrondo, Edward P MitchellAbstract:The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P21 crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P21 has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, β = 105.2°. Model building and refinement are currently under way.
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Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.
Acta crystallographica. Section F Structural biology and crystallization communications, 2006Co-Authors: D Aragão, A R Marques, Francisco J. Enguita, Maria Arménia Carrondo, C Frazão, A M Fialho, I Sá-correia, E P MitchellAbstract:The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P2(1) crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P2(1) has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, beta = 105.2 degrees . Model building and refinement are currently under way.
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Proteins Encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG Genes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities
Applied and environmental microbiology, 2005Co-Authors: Elisabete Silva, Ana Teresa Granja, Arsenio M Fialho, A R Marques, Isabel CorreiaAbstract:The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 μM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 μM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).