Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase

Luca, Claudio De; Lansing, Manfred; Crescenzi, Fabiana; Martini, Irene; Shen, Gwo‐Jenn; O’Regan, Michael H.; Wong, Chi‐Huey

doi:10.1016/0968-0896(95)00159-x

Cited by 19 publications

(10 citation statements)

References 24 publications

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“…The only other bacterial UDP-glucose dehydrogenase that has been purified is that from E. coli (2)(3)(4). Many of the properties of the streptococcal enzyme are similar to that of the E. coli enzyme, with the notable exceptions that the E. coli enzyme is reported to be active as a dimer and that the UDPglucuronic acid inhibition patterns display positive cooperativity.…”

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confidence: 99%

“…It belongs to a small group of dehydrogenases that are able to carry out the 2-fold oxidation of an alcohol to an acid without the release of an aldehyde intermediate (1). Much of the work to date has focused on the properties and mechanism of the beef liver enzyme, and relatively little is known about the enzyme purified from bacterial sources (2)(3)(4). In many strains of bacteria that act as human pathogens, UDPGDH provides the UDP-glucuronic acid required for the construction of an antiphagocytic capsular polysaccharide.…”

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confidence: 99%

“…Many of the known strains of Streptococcus pneumoniae also use UDP-glucuronic acid in the construction of their polysaccharide capsule (11), and it has recently been shown that UDPGDH is required for capsule production in S. pneumoniae type 3 (12). The encapsulated Escherichia coli K5 is also known to use the enzyme for a similar purpose (2,13). The hasB gene that encodes for UDPGDH in group A streptococci (Streptococcus pyogenes) has been cloned and overexpressed in Escherichia coli (14).…”

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Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

Campbell

Sala

Rijn

et al. 1997

Journal of Biological Chemistry

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UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD ؉ -dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDPglucuronate is released last. UDP-xylose was found to be a competitive inhibitor (K i , 2.7 M) of the enzyme. The enzyme is irreversibly inactivated by uridine 5 -diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (k i /K i ) was found to be 2 ؋ 10 3 mM ؊1 min ؊1 . Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5 -diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.The enzyme UDP-glucose dehydrogenase (UDPGDH) 1 catalyzes the NAD ϩ -dependent oxidation of UDP-glucose to UDPglucuronic acid (Fig. 1). It belongs to a small group of dehydrogenases that are able to carry out the 2-fold oxidation of an alcohol to an acid without the release of an aldehyde intermediate (1). Much of the work to date has focused on the properties and mechanism of the beef liver enzyme, and relatively little is known about the enzyme purified from bacterial sources (2-4). In many strains of bacteria that act as human pathogens, UDPGDH provides the UDP-glucuronic acid required for the construction of an antiphagocytic capsular polysaccharide. It is well established that the formation of the capsule is required for virulence (5, 6), and it is thought that the capsule enables the bacteria to evade the host's immune system (7,8). Group A and C streptococci are mammalian pathogens that use UDPGDH in the synthesis of a capsule composed of hyaluronic acid (a polysaccharide consisting of alternating glucuronic acid and N-acetylglucosamine residues) (9, 10). Many of the known strains of Streptococcus pneumoniae also use UDP-glucuronic acid in the construction of their polysaccharide capsule (11), and it has recently been shown that UDPGDH is required for capsule production in S. pneumoniae type 3 (12). The encapsulated Escherichia coli K5 is also known to use the enzyme for a similar purpose (2, 13). The hasB gene that encodes for UDPGDH in group A streptococci (Streptococcus pyogenes) has been cloned and overexpressed in Escherichia coli (14). Its gene product shares 57% sequence identity and 74% sequence similarity with the S. pneumoniae enzyme (15, 16). These properties make th...

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Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

Campbell

Sala

Rijn

et al. 1997

Journal of Biological Chemistry

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“…These results are similar to those of Pattabiraman and Bachhawat, 21 who reported activity over a great range of pH, with the optimum around 8.0, for the enzyme extracted from the sheep brain. Szumilo et al 22 found an optimal pH of around 8.5 for pyrophosphorylase extracted from pig liver, De Luca et al 19 showed a maximum of activity between pH 8-9, and Strominger and Smith 23 reported a pH optimum of 7.2 for the enzyme derived from Staphylococcus aureus. …”

Section: Purification Of Pyrophosphorylasementioning

confidence: 99%

“…Only a few studies have previously reported purification of pyrophosphorylase. De Luca et al 19 purified the cloned enzyme and obtained one purification factor of 46.8. Bulik et al 20 obtained one purification factor of 170 for the pyrophosphorylase from Giardia intestinalis.…”

Section: Purification Of Pyrophosphorylasementioning

confidence: 99%

Purification, characterization and structural determination of UDP-N-acetylglucosamine pyrophosphorylase produced by Moniliophthora perniciosa

Santos¹,

Gonçalves²,

Taranto³

et al. 2011

J. Braz. Chem. Soc.

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A enzima UDP-N-acetilglicosamina pirofosforilase de Moniliophthora perniciosa (CCMB 0257), o fungo patogênico causador da doença vassoura-de-bruxa do Theobroma cacao, foi parcialmente purificada por precipitação com sulfato de amônio e cromatografia de gel filtração em Sephacryl S-200. O tampão de extração da enzima foi o fosfato de sódio, 0,050 mol L -1 , pH 7,0, contendo 1,0 mol L -1 de NaCl. A metodologia de superfície de resposta (MSR) foi usada para a obtenção do pH e temperatura ótima. Os resultados mostraram quatro diferentes isoenzimas (PyroMp I, PyroMp II, PyroMp III e PyroMp IV) que apresentaram pH ótimo na faixa de 6,9-8,4 e temperatura ótima variando entre 28 a 68 °C. A estrutura 3D de pirofosforilase de M. perniciosa foi obtida por modelagem comparativa. O modelo obtido mostrou uma boa qualidade, possuindo 78,6% de aminoácidos nas regiões energeticamente favoráveis. O modelo foi então submetido a simulações de dinâmica molecular (DM). O modelo apresentou uma boa qualidade geométrica após as simulações de DM (91,1% -gráfico de Ramachandran). A procura pelo sítio ativo da enzima mostrou que este é mantido extremamente conservado. Este modelo pode ser útil para desenvolvimento de inibidores contra a doença vassoura de bruxa.The enzyme UDP-N-acetylglucosamine pyrophosphorylase (PyroMp) from Moniliophthora perniciosa (CCMB 0257), a pathogenic fungal strain and the causative agent of the witches' broom disease in Theobroma cacao, was partially purified by precipitation with ammonium sulfate and gel filtration on Sephacryl S-200. The buffer for enzyme extraction was sodium phosphate, 0.050 mol L -1 , pH 7.0, containing 1.0 mol L -1 NaCl. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes (PyroMp I, PyroMp II, PyroMp III and PyroMp IV) were obtained with optimal pH ranging from 6.9-8.4 and optimum temperature ranging from 28 to 68 °C. The 3D structure of pyrophosphorylase of M. perniciosa was determined by comparative modeling. The model obtained showed a good quality, possessing 78.6% of amino acids in energetically allowed regions. The model was then submitted for DM simulation and showed a good geometric quality (91.1% Ramachandran plot). The active site of the enzyme was found to be extremely well conserved. This model will be useful for developing new inhibitors against witches' broom disease.Keywords: pyrophosphorylase, Moniliophthora perniciosa, kinetic characterization, heat stability, 3D structure, comparative modeling J. Braz. Chem. Soc. 1016 Purification, Characterization and Structural Determination of UDP-N-Acetylglucosamine Pyrophosphorylase IntroductionMoniliophthora perniciosa, the causative agent of the witches broom disease in Theobroma cacao, is responsible for major crop losses in South American and Caribbean cocoa plantations. 1,2 In 1989, witches broom disease of cocoa was identified in Bahia, the leading cocoa-growing region in Brazil. 2 In less than 10 years, production shrank from 383,000 tons to an estim...

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Regioselective Benzylation of 2‐Deoxy‐2‐aminosugars using Crown Ethers: Application to a Shortened Synthesis of Hyaluronic Acid Oligomers

2014

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The combination of benzyl bromide, sodium hydroxide and 15-crown-5 in tetrahydrofuran is shown to be an efficient method for installing benzyl groups at both the 4- and 6-positions regioselectively directly from peracetylated N-trichloroacetyl-protected glucosamine and galactosamine. Application of this benzylation strategy proved to significantly shorten the synthetic route to hyaluronic acid tetra- and hexamers.

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Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase

Cited by 19 publications

References 24 publications

Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

Purification, characterization and structural determination of UDP-N-acetylglucosamine pyrophosphorylase produced by Moniliophthora perniciosa

Regioselective Benzylation of 2‐Deoxy‐2‐aminosugars using Crown Ethers: Application to a Shortened Synthesis of Hyaluronic Acid Oligomers

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