Comparative Analysis of the Physiological and Structural Properties of a Medium Chain Length Polyhydroxyalkanoate Depolymerase from Pseudomonas putida KT2442

9 páginas, 6 figuras -- PAGS nros. 1-9Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) produced by Pseudomonas putida KT2442 can be chiral 3-hydroxyalkanoic acids catabolized by the intracellular PhaZ depolymerase when the bacteria are cultured under carbon limitation. PhaZ is an intracellular depolymerase and is attached to PHA granules. In this study, the PhaZ expression was analyzed by Western blot and Real Time RT-PCR assays. The PhaZ expression depends on the carbon source and nitrogen limitation conditions. The substrate recognition of the binding surface was altered using directed mutagenesis experiments. A structural comparison between intracellular and extracellular PHA depolymerases was carried out using PhaZ from P. putida KT2442 and P. fluorescens GK13, respectively. Both share the presence of a core displaying an α/β-hydrolase fold, but the former would possess an additional “lid” domain that suggests some kind of interfacial activation and that may account for differences in substrate specificity for both proteinsThis work was supported by Comunidad Autóno- ma de Madrid Grant AMB-259-0505, by Comisión Interministerial de Ciencia y Tecnología Grants BIO2007-67304-C02 and CTM2006-04007, and by European Union Grants GEN 2006-27750-C5-3-E and NMP2-CT-2007-026515Peer reviewe