Insight into the structure of an endopolygalacturonase from the phytopathogen Burkholderia cepacia: a biochemical and...

We have recently investigated and characterized the mode of action of BcPeh28A, an endopolygalacturonase (endoPG) from the phytopathogen Burkholderia cepacia. EndoPGs belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. Here we report a 3-D structural model of BcPeh28A by combining mass spectrometry (MS) analysis, aimed at disulphide bridges mapping, and computational modelling tools. MS analyses have revealed the complete pattern of disulphide bridges in BcPeh28A, pointing out the presence of three disulphide bonds, defined as Cys3-25, Cys216-244 and Cys309-421. A 3-D model of BcPeh28A was generated by computational methods based on profile-profile sequence alignments and fold recognition algorithms. The final model exhibits a right-handed β-helix fold with eleven β-helical coils and includes the disulphide bonds as additional spatial restraints. Molecular dynamics simulations have been performed to test the conformational stability of the model. Finally, the structural analysis of the BcPeh28A model allows defining the architecture and the amino acid topology of the subsites involved in the catalysis and in the substrate binding specificity.