scholarly journals Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues

2003 ◽  
Vol 374 (2) ◽  
pp. 413-421 ◽  
Author(s):  
Amro A. AMARA ◽  
Bernd H. A. REHM
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Chain Length ◽  
Class Ii ◽  
Base Catalyst ◽  
Wild Type ◽  
General Base ◽  
Medium Chain Length

The class II PHA (polyhydroxyalkanoate) synthases [PHAMCL synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHAMCLs using CoA thioesters of medium-chain-length 3-hydroxyfatty acids (C6–C14) as a substrate. Only recently PHAMCL synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHAMCL synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the α/β-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the α/β-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved γ-turns of the α/β-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the α/β-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.

scholarly journals Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

2013 ◽  
Vol 79 (12) ◽  
pp. 3813-3821 ◽  
Author(s):  
Jo-Ann Chuah ◽  
Satoshi Tomizawa ◽  
Miwa Yamada ◽  
Takeharu Tsuge ◽  
Yoshiharu Doi ◽  
...  
Keyword(s):  
Chain Length ◽  
Fold Increase ◽  
Short Chain ◽  
Pha Synthase ◽  
Wild Type ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

Histone Deacetylase Inhibitors Induce Immuno-Dominant Suppression of Dendritic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 456-456 ◽  
Author(s):  
Pavan Reddy ◽  
Yoshinobu Maeda ◽  
Raimon Duran-Struuck ◽  
Oleg Krijanovski ◽  
Charles Dinarello ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Hdac Inhibitors ◽  
Class Ii ◽  
Wild Type ◽  
Tnf Α

Abstract We and others have recently demonstrated that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor with anti-neoplastic properties, reduces experimental acute graft-versus-host disease (GVHD). We have now investigated the mechanisms of action of two HDAC inhibitors, SAHA and ITF 2357, on allogeneic immune responses. Bone marrow derived dendritic cells (DCs) were preincubated with the HDAC inhibitors at nanomolar concentrations for 16–18 hours and stimulated with lipopolysaccharide (LPS). Pretreatment of DCs caused a significant reduction in the secretion of TNF-α, IL-12p70 and IL-6 compared to the untreated controls (P< 0.005). Similar effects were seen using human peripheral blood mononuclear cell derived DCs. Pre-treatment of both murine and human DCs also significantly reduced their in vitro stimulation of allogeneic T cells as measured by proliferation and IFN-γ production (P<0.01). We determined the in vivo relevance of these observations utilizing a mouse model where the responses of allogeneic donor bm12 T cells depended on the function of injected host B6 DCs would stimulate. Recipient Class-II −/− B6 (H-2b) received 11 Gy on day -1 and were injected with 4–5 x 106 wild type B6 DCs treated with SAHA or with media on days -1 and 0 and then transplanted with 2 x 106 T cells and 5 x 106 TCDBM cells from either syngeneic B6 or allogeneic bm12 donors. SAHA treatment of DCs significantly reduced expansion of allogeneic donor CD4+ T cells on day +7 after BMT compared to controls (P<0.05). SAHA treatment induced a similarly significant reduction in the expansion of CD8+ cells in Class I disparate [bm1→β2M−/−] model. In vitro, SAHA treatment significantly suppressed the expression of CD40 and CD80 but did not alter MHC class II expression. Surprisingly, when mixed with normal DCs at 1:1 ratio, SAHA treated DCs dominantly suppressed allogeneic T cell responses. The regulation of T cell proliferation was not reversible by addition of IL-12, TNF-α, IL-18, anti-IL-10 or anti-TGFβ, either alone or in combination. Suppression of allogeneic responses was contact dependent in trans-well experiments. To address whether the regulation of SAHA treated DCs required contact with T cells, we devised a three cell experiment where SAHA treated DCs lacked the capacity to present antigens to T cells. DCs from B6 MHC Class II deficient (H-2b) were treated with SAHA and co-cultured with wild type B6 (H-2b) DCs along with purified allogeneic BALB/c (H-2d) CD4+ T cells in an MLR. Allogeneic CD4+ T cells proliferated well, demonstrating the regulation to be dependent on contact between SAHA treated DCs and T cells. To address the in vivo relevance of this suppression, we utilized a well characterized [BALB/c →B6] mouse model of acute GVHD. Recipient B6 animals received 11Gy on day -1 and were injected with of 5 million host type SAHA treated or control DCs on days −1, 0, and +2. Mice were transplanted on day 0 with 2 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Injection of SAHA treated DCs resulted in significantly better survival (60% vs. 10%, P < 0.01) and significantly reduced serum levels of TNF-α, donor T cell expansion and histopathology of GVHD on day +7 after BMT compared to the controls. We conclue that HDAC inhibitors are novel immunomodulators that regulate DC function and might represent a novel strategy to prevent GVHD.

scholarly journals Mutagenesis and Chemical Cross-Linking Suggest that Wzz Dimer Stability and Oligomerization Affect Lipopolysaccharide O-Antigen Modal Chain Length Control

2010 ◽  
Vol 192 (13) ◽  
pp. 3385-3393 ◽  
Author(s):  
Magdalene Papadopoulos ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Three Dimensional ◽  
Class Ii ◽  
Cross Linking ◽  
Wild Type ◽  
Class V ◽  
O Antigen ◽  
Class Iv

ABSTRACT In Shigella flexneri, the polysaccharide copolymerase (PCP) protein WzzSF confers a modal length of 10 to 17 repeat units (RUs) to the O-antigen (Oag) component of lipopolysaccharide (LPS). PCPs form oligomeric structures believed to be related to their function. To identify functionally important regions within WzzSF, random in-frame linker mutagenesis was used to create mutants with 5-amino-acid insertions (termed Wzzi proteins), and DNA sequencing was used to locate the insertions. Analysis of the resulting LPS conferred by Wzzi proteins identified five mutant classes. The class I mutants were inactive, resulting in nonregulated LPS Oag chains, while classes II and III conferred shorter LPS Oag chains of 2 to 10 and 8 to 14 RUs, respectively. Class IV mutants retained near-wild-type function, and class V mutants increased the LPS Oag chain length to 16 to 25 RUs. In vivo formaldehyde cross-linking indicated class V mutants readily formed high-molecular-mass oligomers; however, class II and III Wzzi mutants were not effectively cross-linked. Wzz dimer stability was also investigated by heating cross-linked oligomers at 100°C in the presence of SDS. Unlike the WzzSF wild type and class IV and V Wzzi mutants, the class II and III mutant dimers were not detectable. The location of each insertion was mapped onto available PCP three-dimensional (3D) structures, revealing that class V mutations were most likely located within the inner cavity of the PCP oligomer. These data suggest that the ability to produce stable dimers may be important in determining Oag modal chain length.

scholarly journals Hyperencapsulated mucoid pneumococcal isolates from patients with cystic fibrosis have increased biofilm density and persistence in vivo

2018 ◽  
Vol 76 (7) ◽  
Author(s):  
Evida A Dennis ◽  
Mamie T Coats ◽  
Sarah Griffin ◽  
Bing Pang ◽  
David E Briles ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Continuous Flow ◽  
Biofilm Formation ◽  
Capsular Polysaccharides ◽  
Wild Type ◽  
Pneumococcal Colonization ◽  
Phase Variants

AbstractMucoid bacteria, predominately Pseudomonas aeruginosa, are commonly associated with decline in pulmonary function in children with cystic fibrosis (CF), and are thought to persist at least in part due to a greater propensity toward forming biofilms. We isolated a higher frequency of mucoid Streptococcus pneumoniae (Sp) expressing high levels of capsular polysaccharides from sputa from children with CF, compared to those without CF. We compared biofilm formation and maturation by mucoid and non-mucoid isolates of Sp collected from children with and without CF. Non-mucoid Sp serotype 19A and 19F isolates had significantly higher levels of biofilm initiation and adherence to CF epithelial cells than did serotype 3 isolates. However, strains expressing high levels of capsule had significantly greater biofilm maturation, as evidenced by increased density and thickness in static and continuous flow assays via confocal microscopy. Finally, using a serotype 3 Sp strain, we showed that highly encapsulated mucoid phase variants predominate during late adherence and better colonize CFTR–/– as compared to wild-type mice in respiratory infection studies. These findings indicate that overexpression of capsule can enhance the development of mature pneumococcal biofilms in vitro, and may contribute to pneumococcal colonization in CF lung disease.

scholarly journals PchC Thioesterase Optimizes Nonribosomal Biosynthesis of the Peptide Siderophore Pyochelin in Pseudomonas aeruginosa

2004 ◽  
Vol 186 (19) ◽  
pp. 6367-6373 ◽  
Author(s):  
Cornelia Reimmann ◽  
Hiten M. Patel ◽  
Christopher T. Walsh ◽  
Dieter Haas
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Deletion Mutant ◽  
Wild Type ◽  
Peptide Synthetases ◽  
Negative Effect ◽  
In The Wild ◽  
Editing Enzyme ◽  
Maximal Production

ABSTRACT In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.

scholarly journals Pseudomonas aeruginosa Gene Products PilT and PilU Are Required for Cytotoxicity In Vitro and Virulence in a Mouse Model of Acute Pneumonia

1999 ◽  
Vol 67 (7) ◽  
pp. 3625-3630 ◽  
Author(s):  
James C. Comolli ◽  
Alan R. Hauser ◽  
Leslie Waite ◽  
Cynthia B. Whitchurch ◽  
John S. Mattick ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Mouse Model ◽  
Cell Lines ◽  
Twitching Motility ◽  
Wild Type ◽  
Epithelial Cell Lines ◽  
Acute Pneumonia

ABSTRACT Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. ThepilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant. In a mouse model of acute pneumonia, the pilT and pilUmutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

scholarly journals Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

2005 ◽  
Vol 25 (11) ◽  
pp. 4442-4454 ◽  
Author(s):  
Doreen D. Cunningham ◽  
Kathleen Collins
Keyword(s):  
Enzyme Activity ◽  
Telomere Length ◽  
Affinity Purification ◽  
In Vitro Activity ◽  
Wild Type ◽  
Cell Extracts ◽  
Simple Sequence ◽  
Telomere Length Homeostasis

ABSTRACT Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.

Improved Bioavailability of Oxcarbazepine, a BCS class II drug by Centrifugal Melt Spinning: In-vitro and in-vivo Implications

2021 ◽  
pp. 120775
Author(s):  
Sidra Nasir ◽  
Amjad Hussain ◽  
Nasir Abbas ◽  
Nadeem Irfan Bukhari ◽  
Fahad Hussain ◽  
...  
Keyword(s):  
Melt Spinning ◽  
Class Ii ◽  
Bcs Class Ii

scholarly journals Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation

2020 ◽  
Vol 22 (1) ◽  
pp. 152
Author(s):  
Dorota Dabrowska ◽  
Justyna Mozejko-Ciesielska ◽  
Tomasz Pokój ◽  
Slawomir Ciesielski
Keyword(s):  
Chain Length ◽  
Nitrogen Limitation ◽  
Stringent Response ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440 ◽  
Medium Chain ◽  
Environmental Stimuli ◽  
Medium Chain Length ◽  
In The Wild

Pseudomonas putida’s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs’ metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

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