title: Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules.
abstract: A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3( Rru )) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3( Rru ) turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3( Rru )with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3( Rru ) was specific for short-chain-length polyhydroxyalkanoates (PHA(SCL)) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3( Rru ). Low concentrations of calcium or magnesium ions (1-5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3( Rru ) is the representative of a new type of the growing number of intracellular PHB depolymerases.
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Sznajder
authors.forename: Dieter
authors.initials: D
authors.lastname: Jendrossek
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase; D020155:3-Hydroxybutyric Acid