Effects of mutation at position 285 of Ralstonia pickettii T1 poly[(R)-3-hydroxybutyrate] depolymerase on its...

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 is crucial for ester bond cleavage.
• Previous studies identified Asparagine at position 285 (N285) as a key residue.

Purpose of the Study:

• To comprehensively investigate the functional impact of mutations at the N285 position of PHB depolymerase.
• To elucidate the role of N285 in both PHB degradation and p-nitrophenyl ester (pNPCn) hydrolysis.

Main Methods:

• Site-directed saturation mutagenesis was employed to generate N285X mutants.
• Enzyme kinetics were analyzed for pNPCn hydrolysis and PHB degradation.

Main Results:

• Mutant enzymes (N285X) exhibited a reciprocal relationship between PHB-degrading and pNPCn-hydrolyzing activities.
• Histidine (His) substitution at N285 functionally supported PHB degradation, unlike other mutations.

Conclusions:

• The N285 residue plays a critical, albeit complex, role in the catalytic mechanism of PHB depolymerase.
• Specific substitutions, such as Histidine, can retain PHB degradation capabilities, offering insights for enzyme engineering.