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

Manoelito C Santos Junior,Alex G Taranto,Cristiano V Dias,Júlio C M Cascardo,Aristóteles Góes-Neto,Gonçalo A G Pereira,Russolina B Zingali,Carlos P Pirovani,Maria G B Koblitz,Sandra A De Assis,Priscila A Gonçalves,Sandra H Da Cruz

doi:10.1590/s0103-50532011000600003

Abstract

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.

Highlights

Moniliophthora 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]
Pyrophosphorylase was extracted from M. perniciosa as described in Experimental
The purification consisted of only two steps: ammonium sulfate precipitation and gel filtration

Summary

Introduction

Moniliophthora 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]. The 3D structure of pyrophosphorylase of M. perniciosa was determined by comparative modeling.

Results

Conclusion