Tyrosine 105 of Paucimonas lemoignei PHB depolymerase PhaZ7 is essential for polymer binding

Hermawan, S.; Jendrossek, Dieter

doi:10.1016/j.polymdegradstab.2010.01.002

Cited by 8 publications

(8 citation statements)

References 26 publications

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“…The analysis of PhaZ Sa secondary structure (Table 2) supports an alpha/beta hydrolase fold, present in many hydrolytic enzymes such as lipases. A catalytic domain type 1 according to the position of the putative oxyanion hole histidine (closer to the N-terminus than the catalytic amino acids) has been identified, as well as a substrate binding/recognizing domain type 1, with some clearly conserved amino acids (Figure 1) that might play an important role in the establishment of hydrophilic, hydrophobic or electrostatic interactions with the polymeric substrate [41], [42]. The catalytic triad (Ser 131 -Asp 209 -His 269 ) which was previously postulated by multiple sequence alignment [34], is also present, as well as a lipase box pentapeptide (Gly-Leu-Ser-Ala-Gly), which includes the catalytic serine (Ser 131 ).…”

Section: Discussionmentioning

confidence: 99%

Novel Extracellular PHB Depolymerase from Streptomyces ascomycinicus: PHB Copolymers Degradation in Acidic Conditions

et al. 2013

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The ascomycin-producer strain Streptomyces ascomycinicus has been proven to be an extracellular poly(R)-3-hydroxybutyrate (PHB) degrader. The fkbU gene, encoding a PHB depolymerase (PhaZSa), has been cloned in E. coli and Rhodococcus sp. T104 strains for gene expression. Gram-positive host Rhodococcus sp. T104 was able to produce and secrete to the extracellular medium an active protein form. PhaZSa was purified by two hydrophobic interaction chromatographic steps, and afterwards was biochemically as well as structurally characterized. The enzyme was found to be a monomer with a molecular mass of 48.4 kDa, and displayed highest activity at 45°C and pH 6, thus being the first PHB depolymerase from a gram-positive bacterium presenting an acidic pH optimum. The PHB depolymerase activity of PhaZSa was increased in the presence of divalent cations due to non-essential activation, and also in the presence of methyl-β-cyclodextrin and PEG 3350. Protein structure was analyzed, revealing a globular shape with an alpha-beta hydrolase fold. The amino acids comprising the catalytic triad, Ser131-Asp209-His269, were identified by multiple sequence alignment, chemical modification of amino acids and site-directed mutagenesis. These structural results supported the proposal of a three-dimensional model for this depolymerase. PhaZSa was able to degrade PHB, but also demonstrated its ability to degrade films made of PHB, PHBV copolymers and a blend of PHB and starch (7∶3 proportion wt/wt). The features shown by PhaZSa make it an interesting candidate for industrial applications involving PHB degradation.

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Section: Discussionmentioning

confidence: 99%

Novel Extracellular PHB Depolymerase from Streptomyces ascomycinicus: PHB Copolymers Degradation in Acidic Conditions

et al. 2013

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“…Dylight 549 donkey anti rabbit (Cat #711-505-152), Dylight 659 anti-mouse (Cat # 115-495-003), and Dylight 549 anti-mouse (Cat#715–505–150) conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Native polyhydroxybutyrate depolymerase 7, PhaZ7 WT, and a point mutant form of the depolymerase, PhaZ7 S136A, with no enzymatic activity were expressed in E. coli and were purified from the supernatants with ion-affinity chromatography as described in [19] (clones were kindly provided by Dr. D. Jendrossek ).…”

Section: Experimental Partmentioning

confidence: 99%

Identification of the Polyhydroxybutyrate Granules in Mammalian Cultured Cells

Elustondo¹,

Zakharian²,

Pavlov³

2012

Chemistry & Biodiversity

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Poly-3-hydroxybutyrate (PHB) is a biological polyester present in bacteria and eukaryotic cells. Long-chain (or storage) sPHB (up to 100,000 residues) is typically present in PHB-accumulating bacteria and localized in specialized granules known as carbonosomes. In these organisms, sPHB plays a major role as carbon and energy storage. On the other hand, short-chain (or complexed) cPHB (10–100 residues) is present in eukaryotic organisms, including mammals as well as in many bacteria. Previous studies indicated that cPHB is localized in various subcellular compartments of the eukaryotic organisms. Here, we used fluorescent microscopy to directly investigate the localization of PHB in mammalian cells. PHB was visualized in cultured U87 cells using fluorescent probe BODIPY 493/503. Specificity of PHB staining was confirmed by markedly decreased fluorescence of samples treated with PHB-specific depolymerase (PhaZ7). We found that PHB is associated with granules, and that these PHB-enriched granules do not co-localized with mitochondria, lysosomes, or endoplasmic reticulum. These results suggest that, in mammalian cells, PHB can accumulate in the cytoplasm in granules similar to ‘energy storage’ carbonosomes found in PHB-accumulating bacteria.

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“…It is noteworthy that PHB-depolymerase, PhaZ7, is a serine hydrolase with Ser136 as the central amino acid in the catalytic triad, and a single mutation of this serine completely inactivates the enzyme (Braaz et al, 2003). However, the binding of PhaZ7 to its substrate, PHB, is mediated by hydrophobic residues, such as Tyr105, so mutation of this amino acid significantly reduces the ability of the enzyme to bind PHB for hydrolysis (Hermawan and Jendrossek, 2010). Analogously, here PHB is recruited by the hydrophobic residues to the 5S-G mutant peptide, which helps retain this mutant function (Figures 2, 5, 6, and 7A), without the formation of a covalent bond (Figures S13A and S13C).…”

Section: Discussionmentioning

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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Tyrosine 105 of Paucimonas lemoignei PHB depolymerase PhaZ7 is essential for polymer binding

Cited by 8 publications

References 26 publications

Novel Extracellular PHB Depolymerase from Streptomyces ascomycinicus: PHB Copolymers Degradation in Acidic Conditions

Novel Extracellular PHB Depolymerase from Streptomyces ascomycinicus: PHB Copolymers Degradation in Acidic Conditions

Identification of the Polyhydroxybutyrate Granules in Mammalian Cultured Cells

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

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