8 research outputs found
Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF
No full textBackground
To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme.
Results
Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala.
Conclusion
MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development
The emerging role for bacteria in lignin degradation and bio-product formation
No full textThe microbial degradation of lignin has been well studied in white-rot and brown-rot fungi, but is much less well studied in bacteria. Recent published work suggests that a range of soil bacteria, often aromatic-degrading bacteria, are able to break down lignin. The enzymology of bacterial lignin breakdown is currently not well understood, but extracellular peroxidase and laccase enzymes appear to be involved. There are also reports of aromatic-degrading bacteria isolated from termite guts, though there are conflicting reports on the ability of termite gut micro-organisms to break down lignin. If biocatalytic routes for lignin breakdown could be developed, then lignin represents a potentially rich source of renewable aromatic chemicals
Simplified novel muraymycin analogues ; using a serine template strategy for linking key pharmacophores
No full textThe present status of antibiotic research requires the urgent invention of novel agents that act on multidrug-resistant bacteria. The World Health Organization has classified antibiotic-resistant bacteria into critical, high and medium priority according to the urgency of need for new antibiotics. Naturally occurring uridine-derived "nucleoside antibiotics" have shown promising activity against numerous priority resistant organisms by inhibiting the transmembrane protein MraY (translocase I), which is yet to be explored in a clinical context. The catalytic activity of MraY is an essential process for bacterial cell viability and growth including that of priority organisms. Muraymycins are one subclass of naturally occurring MraY inhibitors. Despite having potent antibiotic properties, the structural complexity of muraymycins advocates for simplified analogues as potential lead structures. Herein, we report a systematic structure-activity relationship (SAR) study of serine template-linked, simplified muraymycin-type analogues. This preliminary SAR lead study of serine template analogues successfully revealed that the complex structure of naturally occurring muraymycins could be easily simplified to afford bioactive scaffolds against resistant priority organisms. This study will pave the way for the development of novel antibacterial lead compounds based on a simplified serine template. [Abstract copyright: © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Pharmaceutical applications of lignin-derived chemicals and lignin-based materials : linking lignin source and processing with clinical indication
No full textLignocellulosic biomass is one of the most abundant bioresources on Earth. Over recent decades, various valorisation techniques have been developed to produce value-added products from the cellulosic and hemicellulosic fractions of this biomass. Lignin is the third major component accounting for 10–30% (w/w). However, it currently remains a largely unused fraction due to its recalcitrance and complex structure. The increase in the global demand for lignocellulosic biomass, for energy and chemical production, is increasing the amount of waste lignin available. Approaches to date for valorizing this renewable but heterogeneous chemical resource have mainly focused on production of materials and fine chemicals. Greater value could be gained by developing higher value pharmaceutical applications which would help to improve integrated biorefinery economics. In this review, different lignin extraction methods, such as organosolv and ionic liquid, and the properties and potential of the extracted chemical building blocks are first summarized with respect to pharmaceutical use. The review then discusses the many recent advances made regarding the medical or therapeutic potential of lignin-derived materials such as antimicrobial, antiviral, and antitumor compounds and in controlled drug delivery. The aim is to draw out the link between the source and the processing of the biomass and potential clinical applications. We then highlight four key areas for future research if therapeutic applications of lignin-derived products are to become commercially viable. These relate to the availability and processing of lignocellulosic biomass, technologies for the purification of specific compounds, enhancements in process yield, and progression to human clinical trials
Lignocellulose degradation mechanisms across the Tree of Life
No full textOrganisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role
Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere
No full textOxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling
Investigations into the in vitro developmental plasticity of adult mesenchymal stem cells
No full textBone marrow (BM) derived stem cells contribute to the regeneration of diverse adult tissues including heart, liver and brain following BM transplantation. Trans- differentiation is a mechanism proposed to explain how tissue specific stem cells could generate cells of other organs, thus supporting the emerging concept of enhanced adult stem cell plasticity. New studies have demonstrated that spontaneous cell fusion rather than trans-differentiation is the cause of unexpected cell fate changes in vivo. In contrast, several authors have reported that trans-differentiation can occur in vitro in the absence of cell fusion, including the generation of neural derivatives from non-neural tissues. These findings have profound implications for stem cell biology and cell replacement therapy, and as a result require extensive validation. Mesenchymal stem cells (MSCs) nave been isolated from the postnatal BM and more recently many other sites including adipose tissue, skin and placental cord blood. As such these cells have attracted interest as candidates for cell replacement therapies. This interest follows recent observations both in vitro and in transplant studies that these cells are capable of broader differentiation potential beyond those cell lineages associated with the organ in which they reside. The aim of the present thesis was to examine the developmental plasticity of MSCs in vitro including the capacity of these cells to cross lineage boundaries by differentiating into neuro-ectodermal cell derivatives. There are no universally accepted procedures for the prospective isolation of these cells. In the present thesis, procedures for the isolation of MSCs from rat BM and optimal conditions for the propagation of these cells in culture without loss of multipotent differentiation potential and proliferative capacity are first described. Secondly, the response of cultured MSCs with a consistent immunophenotype to defined culture conditions, previously reported to induce neuronal differentiation of MSCs are evaluated. Thirdly, evidence is presented that suggests that previous claims of trans-differentiation and apparent changes in cell phenotype have been incorrectly interpreted. Evidence is provided that MSCs respond to neural cues in vitro with a stress response, which is characterized by aberrant changes in the expression of constitutive neural proteins, an event previously interpreted as trans-differentiation. MSCs do not have the attributes of early or mature neural derivatives and therefore such changes in protein expression do not equate to true neural differentiation. Finally, evidence is presented that demonstrates that MSCs cultured under defined culture conditions release soluble factors that instruct a neurogenic cell fate decision on neural stem cells (NSCs). In addition, these soluble factors also increase neurite outgrowth of Tuj-1+ differentiating cell progeny. These effects may in part explain the therapeutic benefit of MSG transplantation in animal models of CNS lesions
