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1.
Curr Opin Struct Biol ; 10(6): 687-96, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11114506

RESUMO

L-Rhamnose is a deoxy sugar found widely in bacteria and plants. Evidence continues to emerge about its essential role in many pathogenic bacteria. The crystal structures of two of the four enzymes involved in its biosynthetic pathway have been reported and the other two have been submitted for publication. This pathway does not exist in humans, making enzymes of this pathway very attractive targets for therapeutic intervention.


Assuntos
Ramnose/metabolismo , Cristalografia por Raios X , Enzimas/química , Enzimas/metabolismo , Conformação Proteica , Ramnose/biossíntese
2.
J Mol Biol ; 307(1): 283-95, 2001 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-11243820

RESUMO

l-Rhamnose is a 6-deoxyhexose that is found in a variety of different glycoconjugates in the cell walls of pathogenic bacteria. The precursor of l-rhamnose is dTDP-l-rhamnose, which is synthesised from glucose- 1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway requiring four enzymes. Significantly this pathway does not exist in humans and all four enzymes therefore represent potential therapeutic targets. dTDP-D-glucose 4,6-dehydratase (RmlB; EC 4.2.1.46) is the second enzyme in the dTDP-L-rhamnose biosynthetic pathway. The structure of Salmonella enterica serovar Typhimurium RmlB had been determined to 2.47 A resolution with its cofactor NAD(+) bound. The structure has been refined to a crystallographic R-factor of 20.4 % and an R-free value of 24.9 % with good stereochemistry.RmlB functions as a homodimer with monomer association occurring principally through hydrophobic interactions via a four-helix bundle. Each monomer exhibits an alpha/beta structure that can be divided into two domains. The larger N-terminal domain binds the nucleotide cofactor NAD(+) and consists of a seven-stranded beta-sheet surrounded by alpha-helices. The smaller C-terminal domain is responsible for binding the sugar substrate dTDP-d-glucose and contains four beta-strands and six alpha-helices. The two domains meet to form a cavity in the enzyme. The highly conserved active site Tyr(167)XXXLys(171) catalytic couple and the GlyXGlyXXGly motif at the N terminus characterise RmlB as a member of the short-chain dehydrogenase/reductase extended family. The quaternary structure of RmlB and its similarity to a number of other closely related short-chain dehydrogenase/reductase enzymes have enabled us to propose a mechanism of catalysis for this important enzyme.


Assuntos
Hidroliases/química , NAD/química , Salmonella enterica/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Hidroliases/metabolismo , Dados de Sequência Molecular , Açúcares de Nucleosídeo Difosfato/metabolismo , Nucleotídeos/química , Conformação Proteica , Salmonella enterica/metabolismo , Homologia de Sequência de Aminoácidos , Sorotipagem , Nucleotídeos de Timina/metabolismo
3.
Eur J Biochem ; 222(3): 851-9, 1994 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8026496

RESUMO

The delta-subunit was isolated from the purified yeast F1. Partial protein sequences were determined by direct methods. From this information, degenerated primers were constructed. A part of the ATP delta gene was amplified by polymerase chain reaction from yeast genomic DNA. From the amplified DNA sequence, a nondegenerated oligonucleotide probe was constructed to isolate a 2.6-kbp BamHI-EcoRI DNA fragment bearing the whole gene. A 1036-bp DraI fragment was sequenced. A 480-bp open reading frame encoding a 160-amino-acid polypeptide is described. The deduced amino acid sequence is 22 amino acids longer than the mature protein, which is 138 amino acids long with a mass of 14,555 Da. The delta-subunit of Saccharomyces cerevisiae is 21%, 35%, 52% identical and 66%, 61% and 92% similar to the epsilon-subunit of Escherichia coli and the delta-subunits of beef heart and Neurospora crassa, respectively. A null mutant was constructed. The mutation was recessive and dramatically affected mitochondrial DNA stability since the transformed cells were 100% cytoplasmic petite. The double mutant (rho-, ATP delta::URA3) displayed low or no ATPase activity with an unstable catalytic sector, since a polyclonal antibody directed against the beta subunit did not coprecipitate the alpha subunit.


Assuntos
Genes Fúngicos , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Escherichia coli/enzimologia , Amplificação de Genes , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Peso Molecular , Mutação , Neurospora crassa/enzimologia , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , ATPases Translocadoras de Prótons/isolamento & purificação , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Espectrofotometria Ultravioleta , TATA Box
4.
Eur J Biochem ; 245(3): 813-8, 1997 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9183023

RESUMO

In the yeast Saccharomyces cerevisiae, inactivation of the gene encoding the delta subunit of the ATP synthase led to a lack of assembly of the catalytic sector. In addition a slow-growth phenotype was observed on fermentable medium. This alteration appears in strains lacking intact mitochondrial DNA and showing a defect in the assembly of the catalytic sector, such as the yeast strain inactivated in the gene encoding the epsilon subunit. In rho mitochondria having an intact F1, the ion movement resulting from the exchange of ADP formed in the organelle and ATP entering the mitochondrial compartment led to a mitochondrial transmembranous potential delta psi that was sensitive to carboxyactractyloside. This ion movement was dramatically decreased in rho mitochondria lacking the delta subunit and thus the F1 sector, whereas a cell devoid of delta subunit and complemented with a plasmid harboring the ATPdelta gene displayed an assembled F1, a normal generation time and a fully restored mitochondrial potential. This result could be linked to the involvement of the membrane potential delta psi which is indispensible for mitochondrial biogenesis.


Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Proteína rhoB de Ligação ao GTP , Proteína Inibidora de ATPase
5.
Cell Mol Life Sci ; 58(11): 1650-65, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11706991

RESUMO

Carbohydrates are ideally suited for molecular recognition. By varying the stereochemistry of the hydroxyl substituents, the simple six-carbon, six-oxygen pyranose ring can exist as 10 different molecules. With the further addition of simple chemical changes, the potential for generating distinct molecular recognition surfaces far exceeds that of amino acids. This ability to control and change the stereochemistry of the hydroxyl substituents is very important in biology. Epimerases can be found in animals, plants and microorganisms where they participate in important metabolic pathways such as the Leloir pathway, which involves the conversion of galactose to glucose-1-phosphate. Bacterial epimerases are involved in the production of complex carbohydrate polymers that are used in their cell walls and envelopes and are recognised as potential therapeutic targets for the treatment of bacterial infection. Several distinct strategies have evolved to invert or epimerise the hydroxyl substituents on carbohydrates. In this review we group epimerisation by mechanism and discuss in detail the molecular basis for each group. These groups include enzymes which epimerise by a transient keto intermediate, those that rely on a permanent keto group, those that eliminate then add a nucleotide, those that break then reform carbon-carbon bonds and those that linearize and cyclize the pyranose ring. This approach highlights the quite different biochemical processes that underlie what is seemingly a simple reaction. What this review shows is that each position on the carbohydrate can be epimerised and that epimerisation is found in all organisms.


Assuntos
Bactérias/enzimologia , Metabolismo dos Carboidratos , Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Animais , Bactérias/patogenicidade , Carboidratos/química , Humanos , Isomerismo , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Estrutura Quaternária de Proteína , Prótons , Racemases e Epimerases/genética , Difosfato de Uridina/metabolismo
6.
Acta Crystallogr D Biol Crystallogr ; 54(Pt 5): 891-8, 1998 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9757105

RESUMO

The three-dimensional structure of ubiquitin-conjugating enzyme 9 (Ubc9) has been obtained to a resolution of 2.8 A by molecular replacement followed by a combination of automated refinement and graphical intervention. Diffraction data were recorded on a single crystal in space group P43 with cell dimensions a = b = 73.9, c = 42. 9 A. The final model has an R factor of 21.3% for all data to 2.8 A. Only the N-terminal methionine, a two-residue N-terminal extension and a four-residue loop are not located by the final electron-density map. Ubc9 is now known to be the first sumo, a new ubiquitin-like protein, conjugating enzyme and does not conjugate ubiquitin. The structure of Ubc9 shows important differences compared with the structures of known ubiquitin-conjugating enzymes. At the N-terminal helix, the structural and sequence alignments are out of register by one amino acid giving Ubc9 a different recognition surface compared to ubiquitin-conjugating enzymes. This is coupled to a profound change in the electrostatic surface of the molecular face remote from the catalytic site. These differences may be important in recognition of other proteins in the Sumo conjugation pathway. The catalytic cysteine in Ubc9 has a positively charged lip and a negatively charged ridge nearby. Both these features seem confined to sumo-conjugating enzymes, and a sequence alignment of sumo and ubiquitin suggests how these might play a role in sumo/ubiquitin discrimination.


Assuntos
Ligases/química , Conformação Proteica , Enzimas de Conjugação de Ubiquitina , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Humanos , Ligases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Ubiquitinas/química
7.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 2): 222-5, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10666612

RESUMO

dTDP-D-glucose 4,6-dehydratase (RmlB) is the second of four enzymes involved in the dTDP-L-rhamnose pathway and catalyzes the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The ultimate product of the pathway, dTDP-L-rhamnose, is the precursor of L-rhamnose, which is a key component of the cell wall of many pathogenic bacteria. RmlB from Salmonella enterica serovar Typhimurium has been overexpressed and purified, and crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with lithium sulfate as precipitant. Diffraction data have been obtained to a resolution of 2.8 A on a single frozen RmlB crystal which belongs to space group P2(1), with unit-cell parameters a = 111.85, b = 87.77, c = 145.66 A, beta = 131.53 degrees. The asymmetric unit contains four monomers in the form of two RmlB dimers with a solvent content of 62%. A molecular-replacement solution has been obtained and the model is currently being refined.


Assuntos
Hidroliases/química , Hidroliases/metabolismo , Açúcares de Nucleosídeo Difosfato/biossíntese , Salmonella typhimurium/enzimologia , Nucleotídeos de Timina/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Catálise , Cristalização , Escherichia coli/enzimologia , Escherichia coli/genética , Hidroliases/genética , Hidroliases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Salmonella typhimurium/classificação , Difração de Raios X
8.
Nat Struct Biol ; 7(5): 398-402, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10802738

RESUMO

Deoxythymidine diphosphate (dTDP)-L-rhamnose is the precursor of L-rhamnose, a saccharide required for the virulence of some pathogenic bacteria. dTDP-L-rhamnose is synthesized from glucose-1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway involving four distinct enzymes. This pathway does not exist in humans and the enzymes involved in dTDP-L-rhamnose synthesis are potential targets for the design of new therapeutic agents. Here, the crystal structure of dTDP-6-deoxy-D-xylo-4-hexulose 3,5 epimerase (RmlC, EC5.1.3.13) from Salmonella enterica serovar Typhimurium was determined. The third enzyme of the rhamnose biosynthetic pathway, RmlC epimerizes at two carbon centers, the 3 and 5 positions of the sugar ring. The structure was determined by multiwavelength anomalous diffraction to a resolution of 2.17 A. RmlC is a dimer and each monomer is formed mainly from two beta-sheets arranged in a beta-sandwich. The structure of a dTDP-phenol-RmlC complex shows the substrate-binding site to be located between the two beta-sheets; this site is formed from residues of both monomers. Sequence alignments of other RmlC enzymes confirm that this region is very highly conserved. The enzyme is distinct structurally from other epimerases known and thus, is the first example of a new class of carbohydrate epimerase.


Assuntos
Carboidratos Epimerases/química , Carboidratos Epimerases/classificação , Açúcares de Nucleosídeo Difosfato/metabolismo , Salmonella typhimurium/enzimologia , Nucleotídeos de Timina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Carboidratos Epimerases/metabolismo , Sequência Conservada , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Açúcares de Nucleosídeo Difosfato/química , Fenol/química , Fenol/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Nucleotídeos de Timina/química
9.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 11): 1501-4, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11053865

RESUMO

Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria. RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose. RmlA from Pseudomonas aeruginosa has been overexpressed and purified. Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant. Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.


Assuntos
Açúcares de Nucleosídeo Difosfato/biossíntese , Nucleotidiltransferases/isolamento & purificação , Pseudomonas aeruginosa/enzimologia , Nucleotídeos de Timina/biossíntese , Cromatografia Líquida de Alta Pressão , Cristalização , Cristalografia por Raios X , Nucleotidiltransferases/química , Conformação Proteica
10.
Acta Crystallogr D Biol Crystallogr ; 55(Pt 2): 399-402, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10089346

RESUMO

UDP-galactopyranose mutase, the enzyme responsible for the conversion of UDP-galactopyranose to UDP-galactofuranose, has been crystallized in a form suitable for X-ray diffraction studies. UDP-galactofuranose is a key component of mycobacterial cell walls. Crystals of both the native protein and a selenomethionine variant have been grown by the vapour-diffusion method in hanging drops, and diffract to beyond 3.0 A using synchrotron radiation. Equilibration was against a solution of 20%(w/v) polyethylene glycol (4K), 12%(v/v) 2--propanol, 0.1 M HEPES pH 7.6 at 293.5 K. Crystals grow as thin plates of dimensions 0.4 x 0.2 x approximately 0.02 mm. They are monoclinic [corrected], space group P21, with unit-cell dimensions a = 71. 12, b = 58.42, c = 96.38 A, beta = 96.38 degrees. 92% (native) and 94% (selenomethionine) complete data sets have been recorded to 2.9 A (Rmerge = 5.0%) and 3.0 A (Rmerge = 6.9%), respectively. The Matthews coefficient is 2.35 A3 Da-1 for a dimer in the asymmetric unit, the solvent content being 47%. Diffraction data have also been recorded on a putative platinum derivative to 3.5 A.


Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , Transferases Intramoleculares/química , Sequência de Aminoácidos , Cristalografia por Raios X , Transferases Intramoleculares/isolamento & purificação , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
11.
Acta Crystallogr D Biol Crystallogr ; 55(Pt 3): 706-8, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10089454

RESUMO

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precusor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlC was overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method with ammonium sulfate as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.17 A. The crystal belongs to either space group P3121 or P3221, with unit-cell parameters a = b = 71.56, c = 183.53 A and alpha = beta = 90, gamma = 120 degrees.


Assuntos
Carboidratos Epimerases/isolamento & purificação , Ramnose/biossíntese , Salmonella typhimurium/enzimologia , Carboidratos Epimerases/química , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cristalização , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ramnose/análogos & derivados
12.
Acta Crystallogr D Biol Crystallogr ; 55(Pt 12): 2043-6, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10666586

RESUMO

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlD catalyses the terminal step of this pathway by converting dTDP-6-deoxy-L-lyxo-4-hexulose to dTDP-L-rhamnose. RmlD from -Salmonella enterica serovar Typhimurium has been overexpressed in Escherichia coli. The recombinant protein was purified by a two--step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals of native and selenomethionine-enriched RmlD have been obtained using the sitting-drop vapour-diffusion method with polyethylene glycol as precipitant. Diffraction data have been collected from orthorhombic crystals of both native and selenomethionyl-derivatized protein, allowing tracing of the protein structure.


Assuntos
Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/isolamento & purificação , Salmonella typhimurium/enzimologia , Desidrogenases de Carboidrato/genética , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Açúcares de Nucleosídeo Difosfato/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Salmonella typhimurium/genética , Nucleotídeos de Timina/biossíntese
13.
Biochem Soc Trans ; 31(Pt 3): 532-6, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12773151

RESUMO

Bacteria have a rich collection of biochemical pathways for the synthesis of complex metabolites. These conversions often involve chemical reactions that are hard to reproduce in the laboratory. An area of considerable interest is in the manipulation and synthesis of carbohydrates. In contrast with amino acids, carbohydrates are densely functionalized (each carbon atom is attached to at least one heteroatom) and this holds out the prospect of discovering novel enzyme mechanisms. The results from the study of the biosynthetic dTDP-L-rhamnose pathway are discussed. dTDP-L-rhamnose is a key intermediate in many pathogenic bacteria, as it is the donor for L-rhamnose, which is found in the cell wall of important human pathogens, such as Mycobacteria tuberculosis and Salmonella typhimurium. All four enzymes have been structurally characterized; in particular, the acquisition of structural data on substrate complexes was extremely useful. The structural data have guided site-directed-mutagenesis studies that have been used to test mechanistic hypotheses. The results shed light on three classes of enzyme mechanism: nucleotide condensation, short-chain dehydrogenase activity and epimerization.


Assuntos
Glucose/metabolismo , Hidroliases/metabolismo , Açúcares de Nucleosídeo Difosfato/química , Açúcares de Nucleosídeo Difosfato/metabolismo , Nucleotidiltransferases/metabolismo , Ramnose/metabolismo , Hidroliases/química , Modelos Moleculares , Nucleotidiltransferases/química , Conformação Proteica
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