title: The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase.
abstract: A approximately 35 kDa protein that has been described to be secreted by Paucimonas lemoignei during growth on succinate and to inhibit hydrolysis of denatured (crystalline) poly(3-hydroxybutyrate) (dPHB) by extracellular PHB depolymerases of P. lemoignei (PHB depolymerase inhibitor (PDI)) was purified and characterized. Purified PDI (M(r), 36 199 +/- 45 Da) inhibited hydrolysis of dPHB by two selected purified PHB depolymerases (PhaZ2 and PhaZ5) but did not inhibit the hydrolysis of water-soluble substrates such as p-nitrophenylbutyrate by PhaZ5 and PhaZ2. PDI revealed a high binding affinity to dPHB although it was not able to hydrolyze the crystalline polymer. However, purified PDI had a high hydrolytic activity if native (amorphous) PHB (nPHB) was used as a substrate. N-terminal sequencing of PDI revealed that it was identical to recently described extracellular PHB depolymerase PhaZ7 which is specific for nPHB and which cannot hydrolyze dPHB. To confirm that the inhibition of hydrolysis of dPHB by PhaZ7 is an indirect surface competition effect at high depolymerase concentration, the activity of PHB depolymerases PhaZ2 and PhaZ5 in the presence of different amounts of protein mixtures was determined. The components of NB or LB medium inhibited hydrolysis of the polymer in a concentration-dependent manner but had no effect on the hydrolysis of p-nitrophenylbutyrate by PHB depolymerases. In combination with PHB depolymerases PhaZ2 and PhaZ5 the protein PhaZ7 ("PDI") enables the bacteria to hydrolyze dPHB and nPHB simultaneously.
authors.affiliation: Institut für Mikrobiologie, Universität Stuttgart, 70550 Stuttgart, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Reinhardt
authors.forename: René
authors.initials: R
authors.lastname: Handrick
authors.forename: Dieter
authors.initials: D
authors.lastname: Jendrossek
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000494:pharmacology*; D006868:Hydrolysis; D006885:Hydroxybutyrates / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D011091:Polyesters / Q000378:metabolism; D011549:Pseudomonas; D000094122:Polyhydroxybutyrates
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C438011:PHAZ7 protein, Paucimonas lemoignei; C022417:poly-beta-hydroxybutyrate depolymerase