Other PHAC publications
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC10111563 | biostudies-literature | 2023 Apr
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.
| S-EPMC3347165 | biostudies-literature
Project description:Biodegradable polyester polyhydroxyalkanoate (PHA) is a promising bioplastic material for industrial use as a replacement for petroleum-based plastics. PHA synthase PhaC forms an active dimer to polymerize acyl moieties from the substrate acyl-coenzyme A (CoA) into PHA polymers. Here we present the crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, bound to CoA. The structure reveals an asymmetric dimer, in which one protomer adopts an open conformation bound to CoA, whereas the other adopts a closed conformation in a CoA-free form. The open conformation is stabilized by the asymmetric dimerization and enables PhaC to accommodate CoA and also to create the product egress path. The bound CoA molecule has its β-mercaptoethanolamine moiety extended into the active site with the terminal SH group close to active center Cys291, enabling formation of the reaction intermediate by acylation of Cys291.
| S-EPMC7214940 | biostudies-literature
Project description:Citation networks of scientific publications offer fundamental insights into the structure and development of scientific knowledge. We propose a new measure, called intermediacy, for tracing the historical development of scientific knowledge. Given two publications, an older and a more recent one, intermediacy identifies publications that seem to play a major role in the historical development from the older to the more recent publication. The identified publications are important in connecting the older and the more recent publication in the citation network. After providing a formal definition of intermediacy, we study its mathematical properties. We then present two empirical case studies, one tracing historical developments at the interface between the community detection literature and the scientometric literature and one examining the development of the literature on peer review. We show both conceptually and empirically how intermediacy differs from main path analysis, which is the most popular approach for tracing historical developments in citation networks. Main path analysis tends to favour longer paths over shorter ones, whereas intermediacy has the opposite tendency. Compared to the main path analysis, we conclude that intermediacy offers a more principled approach for tracing the historical development of scientific knowledge.
| S-EPMC7029947 | biostudies-literature
Project description:PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short-chain-length (scl) or medium-chain-length (mcl) polymers: Class I, Class III and Class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while Class II PhaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of Class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/Ralstonia eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by X-ray absorption fine-structure (XAFS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation, and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, and for the synthesis of PHB copolymers and medium-chain-length PHA polymers with optimized chemical properties.
| S-EPMC6403647 | biostudies-literature
Project description:Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaC Cs -CAT. The structure shows that PhaC Cs -CAT forms an α/β hydrolase fold comprising α/β core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaC Cn -CAT). Structural comparison showed PhaC Cn -CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaC Cs -CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaC Cn -CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.
| S-EPMC5509742 | biostudies-literature
Project description:Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to the phaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaC(Bm)) and PhaR (PhaR(Bm)) for activity in vivo and in vitro. PhaC(Bm) showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaR(Bm) had no obvious homology to PhaE. Previously we showed that PhaC(Bm), and here we show that PhaR(Bm), is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaC(Bm) to produce an active PHA synthase.
| S-EPMC95313 | biostudies-literature
Project description: Not available
| S-EPMC5833570 | biostudies-literature
Project description:The formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha are controlled by PhaM, which interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance in attaching PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in detachment of formed PHB granules from the nucleoid. Instead, formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells were observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct colocalization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaCC319A protein were not able to form fluorescent foci, indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach from formed PHB granules at later stages, resulting in a nonhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB-permissive conditions at a certain size.IMPORTANCE PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells, and for association of formed PHB granules with the nucleoid. Here we found that four lysine residues of C-terminal PhaM sequence motifs are essential for association of PHB granules with the nucleoid. Furthermore, we followed PHB granule formation by time-lapse microscopy and provide evidence for aging of PHB granules that is manifested by detachment of previously PHB granule-associated PhaM and PHB synthase.
| S-EPMC5452819 | biostudies-literature
Project description:Clinical use of doxorubicin (Dox) is limited by cumulative myelo- and cardiotoxicity. This research focuses on the detailed characterization of PhAc-ALGP-Dox, a targeted tetrapeptide prodrug with a unique dual-step activation mechanism, designed to circumvent Dox-related toxicities and is ready for upcoming clinical investigation. Coupling Dox to a phosphonoacetyl (PhAc)-capped tetrapeptide forms the cell-impermeable, inactive compound, PhAc-ALGP-Dox. After extracellular cleavage by tumor-enriched thimet oligopeptidase-1 (THOP1), a cell-permeable but still biologically inactive dipeptide-conjugate is formed (GP-Dox), which is further processed intracellularly to Dox by fibroblast activation protein-alpha (FAPα) and/or dipeptidyl peptidase-4 (DPP4). In vitro, PhAc-ALGP-Dox is effective in various 2D- and 3D-cancer models, while showing improved safety toward normal epithelium, hematopoietic progenitors, and cardiomyocytes. In vivo, these results translate into a 10-fold higher tolerability and 5-fold greater retention of Dox in the tumor microenvironment compared with the parental drug. PhAc-ALGP-Dox demonstrates 63% to 96% tumor growth inhibition in preclinical models, an 8-fold improvement in efficacy in patient-derived xenograft (PDX) models, and reduced metastatic burden in a murine model of experimental lung metastasis, improving survival by 30%. The current findings highlight the potential clinical benefit of PhAc-ALGP-Dox, a targeted drug-conjugate with broad applicability, favorable tissue biodistribution, significantly improved tolerability, and tumor growth inhibition at primary and metastatic sites in numerous solid tumor models.
| S-EPMC9377749 | biostudies-literature
Project description:Cohort studies collect, generate and distribute data over long periods of time - often over the lifecourse of their participants. It is common for these studies to host a list of publications (which can number many thousands) on their website to demonstrate the impact of the study and facilitate the search of existing research to which the study data has contributed. The ability to search and explore these publication lists varies greatly between studies. We believe a lack of rich search and exploration functionality of study publications is a barrier to entry for new or prospective users of a study's data, since it may be difficult to find and evaluate previous work in a given area. These lists of publications are also typically manually curated, resulting in a lack of rich metadata to analyse, making bibliometric analysis difficult. We present here a software pipeline that aggregates metadata from a variety of third-party providers to power a web based search and exploration tool for lists of publications. Alongside core publication metadata (i.e. author lists, keywords etc.), we include geocoding of first authors and citation counts in our pipeline. This allows a characterisation of a study as a whole based on common locations of authors, frequency of keywords, citation profile etc. This enriched publications metadata can be useful for generating study impact metrics and web-based graphics for public dissemination. In addition, the pipeline produces a research data set for bibliometric analysis or social studies of science. We use a previously published list of publications from a cohort study as an exemplar input data set to show the output and utility of the pipeline here.
| S-EPMC8108552 | biostudies-literature