Y443F mutation in the substrate-binding domain of extracellular PHB depolymerase enhances its PHB adsorption and disruption abilities

Abstract

Extracellular poly[( R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ RpiT1) from Ralstonia pickettii T1 adsorbs to the PHB surface via its substrate-binding domain (SBD) and cleaves the PHB chain using its catalytic domain. Our previous study (Biomacromolecules 2010; 11: 113–119) has suggested that the hydrophobic interaction between the amino acid residues at positions 441, 443, and 445 in the SBD and the PHB surface plays a crucial role in facilitating the association phase of the enzyme adsorption process. In the present study, in order to improve PhaZ RpiT1 for effective PHB degradation, we targeted Tyr at position 443 for substitution with a more highly hydrophobic amino acid residue because its hydrophobicity shows medium to high degree compared to those of general naturally occurring amino acid residues. We designed a mutant enzyme with an amino acid substitution at this position, taking the following factors into consideration: (1) to achieve higher hydrophobicity than the original residue, (2) to retain the β-sheet structure, and (3) to change as little as possible the volume of the amino acid residue after the substitution. As a result, the substitution of Tyr443 with Phe (Y443F) was considered to be appropriate. The purified Y443F enzyme showed identical CD spectrum and hydrolysis activity for a water-soluble substrate with the wild type, indicating that the mutation had no influence on the structure and the ester bond cleavage activity. In contrast, the Y443F enzyme had higher PHB degradation activity than the wild type. Kinetic analysis of PHB degradation suggests that this amino acid substitution promoted not only the adsorption of the mutant enzyme to PHB, but also the disruption of the PHB surface to enhance the hydrolysis of the PHB polymer chain.