The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase.

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.