Effects of Mutations in the Substrate-Binding Domain of Poly[( R )-3-Hydroxybutyrate] (PHB) Depolymerase from Ralstonia pickettii T1 on PHB Degradation

Abstract

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase fromRalstonia pickettii T1 (PhaZ RpiT1 ) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ RpiT1 was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ RpiT1 . Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ RpiT1 adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.Poly[(R)-3-hydroxyalkanoate]s (PHAs) are produced by a wide variety of bacteria and show a remarkable biodegradability in natural environments such as soil (11,19), activated sludge (33), freshwater (20), and seawater (21). Because of their biodegradability and physical properties, which are comparable to those of conventional plastics, PHAs have attracted academic and industrial interest (1,5,9). Poly[(R)-3-hydroxybutyrate] (PHB), which is found in many types of bacteria, exists in different states in the cells and after extraction from the cells. In the cells, PHB forms amorphous granules (native PHB [nPHB]) and can be degraded by intracellular PHB depolymerases, which are produced by the PHB-accumulating bacterium itself. After nPHB is extracted from the cells, nPHB is converted to a semicrystalline form (denatured PHB [dPHB]), which can be enzymatically degraded by extracellular PHB depolymerases (dPHB depolymerases) secreted from various microorganisms in the environment.A number of dPHA depolymerases have been purified and characterized (9, 13), and over 20 dPHA depolymerase genes have been cloned and analyzed (9,15,26). Genetic analyses of the enzymes have shown that dPHB depolymerases consist of a catalytic domain at the N terminus, a substrate-binding domain (SBD) at the C terminus, and a linker region connecting the two domains. The structure-function relationship of dPHB depolymerases has been studied extensively, and se...

Autoregulator Protein PhaR for Biosynthesis of Polyhydroxybutyrate [P(3HB)] Possibly Has Two Separate Domains That Bind to the Target DNA and P(3HB): Functional Mapping of Amino Acid Residues Responsible for DNA Binding

“…In contrast to the high sequence homology in the N-terminal regions among PhaR homologs, sequence similarity is partly distributed in the predicted P(3HB)-binding motif region shared by PhaR family members. The next project has been initiated to address the amino acid residues of PhaR responsible for binding to P(3HB) by two approaches, X-ray crystallographic analysis and further random mutagenesis analysis, as also employed for a member of the granule-associated proteins, P(3HB) depolymerase (10,11).…”

Degradation of Natural and Artificial Poly[(R)-3-hydroxyalkanoate]s: From Biodegradation to Hydrolysis

“…The difference in the crystal structure influenced the enzymatic degradation. Absorption research on R. pickettii T1 depolymerase mutant on PHA revealed that the absorption depended not only on hydrogen bonds between hydroxyl groups of serine in the enzyme and carbonyl groups in the PHB polymer, but also on hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer (Hiraishi et al 2006;Abe et al 2005). The number of PHB depolymerase enzyme molecules adsorbed on each single crystal increased in the following order: poly(3HB-co-8 mol% 3HH) < PHB » poly(3HB-co-6 mol% 3HV), where 3HH is (R)-3-hydroxyhexanoate.…”

Display of Functionally Active PHB Depolymerase on Escherichia Coli Cell Surface

“…The substrate-binding domain is responsible for the enzyme adsorption to the polymer surface as well as the disruption of the polymer surface structure, resulting in the easy access of the catalytic domain to the polymer chains. [9][10][11][12][13][14][15][16] It is possible that such domain structure of PHB depolymerase is responsible for the effective enzymatic degradation of PHB at the solid/liquid interface. Besides its excellent ability to depolymerize the biosynthesized PHB, because PHB depolymerase is derived from renewable resources, this enzyme shows great promise as a low-cost, human-friendly, and environmentally compatible catalyst for PHB monomer production by serving as an alternative to chemical catalysts.…”