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The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase
Simone Reinhardt1, René Handrick, Dieter Jendrossek
1Institut für Mikrobiologie, Universität Stuttgart, 70550 Stuttgart, Germany.
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.
Insights
A protein secreted by Paucimonas lemoignei, identified as PHB depolymerase inhibitor (PDI) and PhaZ7, inhibits crystalline poly(3-hydroxybutyrate) hydrolysis. This PDI/PhaZ7 protein enables simultaneous hydrolysis of both crystalline and amorphous PHB when combined with other depolymerases.
Area of Science:
- Microbiology
- Biochemistry
- Polymer Science
Background:
- Paucimonas lemoignei secretes a protein inhibiting poly(3-hydroxybutyrate) (PHB) hydrolysis.
- This protein, PHB depolymerase inhibitor (PDI), affects extracellular PHB depolymerases.
- PDI was previously described to inhibit crystalline PHB hydrolysis.
Purpose of the Study:
- To purify and characterize the PDI protein.
- To elucidate the mechanism of PDI's inhibition of PHB hydrolysis.
- To investigate the role of PDI in the simultaneous hydrolysis of different PHB forms.
Main Methods:
- Protein purification and characterization (M.W. determination).
- Enzyme activity assays using crystalline PHB (dPHB), amorphous PHB (nPHB), and p-nitrophenylbutyrate.
- N-terminal sequencing to identify the protein.
- Analysis of depolymerase activity in the presence of varying protein concentrations.
Main Results:
- Purified PDI (36,199 Da) inhibited hydrolysis of dPHB by PhaZ2 and PhaZ5 but not soluble substrates.
- PDI showed high affinity for dPHB but could not hydrolyze it; however, it efficiently hydrolyzed nPHB.
- N-terminal sequencing identified PDI as PhaZ7, an extracellular PHB depolymerase specific for nPHB.
- Inhibition of dPHB hydrolysis by PhaZ7 was an indirect surface competition effect at high concentrations.
- PhaZ7, in combination with PhaZ2 and PhaZ5, enabled P. lemoignei to simultaneously hydrolyze both dPHB and nPHB.
Conclusions:
- The PHB depolymerase inhibitor (PDI) is identical to the extracellular PHB depolymerase PhaZ7.
- PhaZ7 inhibits crystalline PHB hydrolysis through surface competition, not direct enzymatic activity.
- PhaZ7 plays a role in enabling bacteria to degrade both crystalline and amorphous PHB forms concurrently.