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

Journal of the Brazilian Chemical Society

Manoelito C. Santos Junior²  Priscila A. Gonçalves²  Alex G. Taranto¹  Maria G. B. Koblitz¹  Aristóteles Góes-neto¹  Carlos P. Pirovani¹  Júlio C. M. Cascardo¹  Sandra H. Da Cruz¹  Russolina B. Zingali¹  Gonçalo A. G. Pereira¹  Cristiano V. Dias¹  Sandra A. De Assis² 

[1] ,Universidade Estadual de Feira de Santana Departamento de Saúde Feira de Santana BA ,Brazil

关键词: pyrophosphorylase;    Moniliophthora perniciosa;    kinetic characterization;    heat stability;    3D structure;    comparative modeling;   

DOI  :  10.1590/S0103-50532011000600003

来源: SciELO

【 摘 要 】

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.

【 授权许可】

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