The “PHB Depolymerase Inhibitor” of Paucimonas lemoignei Is a PHB Depolymerase

Reinhardt, Simone; Handrick, René; Jendrossek, Dieter

doi:10.1021/bm025519x

Cited by 5 publications

(4 citation statements)

References 15 publications

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“…P. lemoignei secretes at least seven different e-PHA depolymerases: PhaZ1-PhaZ7. The first six depolymerases (PhaZ1-PhaZ6) are active against dPHB, whereas PhaZ7 is active against the amorphous form, mainly nPHB (Handrick et al, 2001;Reinhardt et al, 2002). Accordingly, the first six depolymerases are termed e-dPHA depolymerases, whereas PhaZ7 is termed an e-nPHA depolymerase (Jendrossek, 2001).…”

Section: Introductionmentioning

confidence: 99%

The structure of PhaZ7 at atomic (1.2 Å) resolution reveals details of the active site and suggests a substrate-binding mode

et al. 2010

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Poly-(R)-hydroxyalkanoates (PHAs) are bacterial polyesters that are degraded by a group of enzymes known as PHA depolymerases. Paucimonas lemoignei PhaZ7 depolymerase is the only extracellular depolymerase that has been described as being active towards amorphous PHAs. A previously determined crystal structure of PhaZ7 revealed an alpha/beta-hydrolase fold and a Ser-His-Asp catalytic triad. In order to address questions regarding the catalytic mechanism and substrate binding, the atomic resolution structure of PhaZ7 was determined after cocrystallization with the protease inhibitor PMSF. The reported structure has the highest resolution (1.2 A) of currently known depolymerase structures and shows a sulfur dioxide molecule covalently attached to the active-site residue Ser136. Structural comparison with the free PhaZ7 structure (1.45 A resolution) revealed no major changes in the active site, suggesting a preformed catalytic triad. The oxyanion hole was found to be formed by the amide groups of Met137 and Asn49. Nine well ordered water molecules were located in the active site. Manual docking of a substrate trimer showed that the positions of these water molecules coincide well with the substrate atoms. It is proposed that these water molecules are displaced upon binding of the substrate. Furthermore, conformational changes were identified after comparison with a previously determined PhaZ7 dimer structure in a different space group. The changes were located in surface loops involved in dimer formation, indicating some flexibility of these loops and their possible involvement in polyester binding.

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Section: Introductionmentioning

confidence: 99%

The structure of PhaZ7 at atomic (1.2 Å) resolution reveals details of the active site and suggests a substrate-binding mode

et al. 2010

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“…purified from the bacterial strains such as Ralstonia picketti, [4,5] Paucimonas lemoignei, [6][7][8][9][10][11][12] Pseudomonas stutzeri, [13,14] Comamonas testosteroni, [15,16] and Comamonas acidovorans. [17] Characterization of the structural genes has revealed that PHB depolymerases are composed of two domains and a linker region.…”

Section: Introductionmentioning

confidence: 99%

“…PHAs are synthesized and accumulated in a bacterial cell as a carbon and energy storage material by a wide variety of bacteria,1–3 and hydrolyzed by extracellular poly(hydroxybutyrate) (PHB) depolymerases which are secreted from a number of microorganisms. Several extracellular PHB depolymerases have been purified from the bacterial strains such as Ralstonia picketti ,4,5 Paucimonas lemoignei ,6–12 Pseudomonas stutzeri ,13,14 Comamonas testosteroni ,15,16 and Comamonas acidovorans 17…”

Section: Introductionmentioning

confidence: 99%

Characteristic Interactions between Poly(hydroxybutyrate) Depolymerase and Poly[(R)‐3‐hydroxybutyrate] Film Studied by a Quartz Crystal Microbalance

Yamashita

Funato

Suzuki

et al. 2003

Macromolecular Bioscience

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Enzymatic degradation of poly[(R)‐3‐hydroxybutyrate] (P(3HB)) film by the poly(hydroxybutyrate) (PHB) depolymerase from Ralstonia picketti T1 was studied in 0.01 M phosphate buffer solution (pH 7.4) at 37 °C by using a quartz crystal microbalance (QCM) technique. Enzymatic degradation of P(3HB) film was quantitatively followed by QCM as a positive frequency shift. While, the amount of depolymerases adsorbed on the film could be evaluated as a negative frequency shift by using a mutant enzyme which had no hydrolytic activity in a catalytic site. The degradation rate increased with enzyme concentration to reach a maximum value at 1.0 μg · mL−1, and then the rate decreased at higher enzyme concentration. This enzyme concentration dependence could be quantitatively explained in terms of a change of coverage of the film surface by the adsorbed enzyme. When the wild‐type enzyme solution in a QCM cell was replaced with the mutant enzyme solution in the middle of the reaction, the degradation rate was reduced markedly, indicating that the wild‐type enzyme adsorbed on the P(3HB) surface is easily substituted by the mutant enzyme in the solution. On the other hand, replacement of the wild‐type enzyme solution with other proteins or buffer solutions did not affect the degradation rate at all, suggesting that the adsorbed enzyme was not desorbed from the film surface. Thus, the adsorbed PHB depolymerase is released from the P(3HB) surface only by interaction with the same depolymerase in solution.

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“…Alternatively, we studied TRPM8 activity in the presence of the PHB hydrolyzing enzyme, PHB-depolymerase, PhaZ7, a serine-hydrolase family enzyme that is naturally expressed in the bacterium Paucimonas lemoignei (Kapetaniou et al, 2005; Reinhardt et al, 2002). We employed the PhaZ7 enzyme in two different applications.…”

Section: Resultsmentioning

confidence: 99%

Polyester Modification of the Mammalian TRPM8 Channel Protein: Implications for Structure and Function

Cao¹,

Yudin²,

Bikard³

et al. 2013

Cell Reports

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SUMMARY The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors, and is likely to be pre-conditioned by its molecular composition. Our studies indicate that TRPM8 channel forms a structural-functional complex with the polyester, poly-(R)-3hydroxybutyrate (PHB). We identified by mass spectrometry a number of PHB-modified peptides in the N-terminus of the TRPM8 protein and in its extracellular S3–S4 linker. Removal of PHB by enzymatic hydrolysis, and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer, resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes post-translational modification by PHB and that this modification is required for its normal function.

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The “PHB Depolymerase Inhibitor” of Paucimonas lemoignei Is a PHB Depolymerase

Cited by 5 publications

References 15 publications

The structure of PhaZ7 at atomic (1.2 Å) resolution reveals details of the active site and suggests a substrate-binding mode

The structure of PhaZ7 at atomic (1.2 Å) resolution reveals details of the active site and suggests a substrate-binding mode

Characteristic Interactions between Poly(hydroxybutyrate) Depolymerase and Poly[(R)‐3‐hydroxybutyrate] Film Studied by a Quartz Crystal Microbalance

Polyester Modification of the Mammalian TRPM8 Channel Protein: Implications for Structure and Function

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