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Enhanced Clearing of Wound-Related Pathogenic Bacterial Biofilms Using Protease-Functionalized Antibiotic Nanocarriers.

Biofilms are prevalent in chronic wounds and once formed are very hard to remove, which is associated with poor outcomes and high mortality rates. Biofilms are comprised of surface-attached bacteria embedded in an extracellular polymeric substance (EPS) matrix, which confers increased antibiotic resistance and host immune evasion. Therefore, disruption of this matrix is essential to tackle the biofilm-embedded bacteria. Here, we propose a novel nanotechnology to do this, based on protease-functionalized nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with t

Functional characterisation of the methionine sulfoxide reductase repertoire in Trypanosoma brucei.

To combat the deleterious effects that oxidation of the sulfur atom in methionine to sulfoxide may bring, aerobic cells express repair pathways involving methionine sulfoxide reductases (MSRs) to reverse the above reaction. Here, we show that Trypanosoma brucei, the causative agent of African trypanosomiasis, expresses two distinct trypanothione-dependent MSRs that can be distinguished from each other based on sequence, sub-cellular localisation and substrate preference. One enzyme found in the parasite's cytosol, shows homology to the MSRA family of repair proteins and preferentially metaboli

Immunomodulation by the Pseudomonas syringae HopZ type III effector family in Arabidopsis.

Pseudomonas syringae employs a type III secretion system to inject 20-30 different type III effector (T3SE) proteins into plant host cells. A major role of T3SEs is to suppress plant immune responses and promote bacterial infection. The YopJ/HopZ acetyltransferases are a superfamily of T3SEs found in both plant and animal pathogenic bacteria. In P. syringae, this superfamily includes the evolutionarily diverse HopZ1, HopZ2 and HopZ3 alleles. To investigate the roles of the HopZ family in immunomodulation, we generated dexamethasone-inducible T3SE transgenic lines of Arabidopsis for HopZ family

Acetohydroxyacid synthase: a target for antimicrobial drug discovery.

Acetohydroxyacid synthase (AHAS) (EC 2.2.1.6) (also known as acetolactate synthase) is the first common enzyme in the branched chain amino acid (BCAA) biosynthesis pathway. This pathway is present in microorganisms and in plants but not in animals, making it an attractive target for both drug and herbicide discovery. The function of AHAS is to catalyze the conversion of two molecules of pyruvate to 2-acetolactate or to convert one molecule of pyruvate and a molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. Three cofactors are required for the activity of AHAS: thiamine diphosphate (Th

Molecular basis for the antiparasitic activity of a mercaptoacetamide derivative that inhibits histone deacetylase 8 (HDAC8) from the human pathogen schistosoma mansoni.

Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides severa

Production of a robust nanobiocatalyst for municipal wastewater treatment.

Immobilization is a fundamental method to improve both enzyme activity and stability. In the present work, the process previously described for immobilizing laccase - an enzyme oxidizing phenolic compounds - onto fumed silica was optimized, in order to efficiently produce industrially relevant amounts of a nanobiocatalyst for biological micropollutant elimination, whilst saving 80% of surface modification agent (3-aminopropyl triethoxy silane) and 90% of cross-linker (glutaraldehyde). Minimized losses during preparation and favorable effects of immobilization yielded conjugates with drasticall

Cloning, characterization and mutagenesis of Russell's viper venom L-amino acid oxidase: Insights into its catalytic mechanism.

To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic l-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D.

Advanced Technologies for Large Scale Supply of Marine Drugs.

Marine organisms represent a source of unique chemical entities with valuable biomedical potentialities, broad diversity, and complexity. It is essential to ensure a reliable and sustainable supply of marine natural products (MNPs) for their translation into commercial drugs and other valuable products. From a structural point of view and with few exceptions, MNPs of pharmaceutical importance derive from the so-called secondary metabolism of marine organisms. When production strategies rely on marine macroorganisms, harvesting or culturing coupled with extraction procedures frequently remain t

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… These are some representative examples where the use of biocatalysis in conjunction with synthetic chemistry presents a promising strategy for the efficient production of therapeutics, particularly in the context of marine natural products. … Complications with enzyme supply, labor-intensive protein purification, substrate solubility in aqueous buffer, and cofactor expense can all be hurdles to preparative-scale biocatalysis. 5. …

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down t

Selective peroxynitrite-mediated protein nitration catalyzed by glyoxalase domain containing protein 4.

Tyrosine nitration alters the structure, function, and cellular localization of proteins and is implicated in the pathology of multiple diseases [G. Ferrer-Sueta et al., Chem. Rev. 118, 1338-1408 (2018), H. Ischiropoulos, Arch. Biochem. Biophys. 356, 1-11 (1998), I. Griswold-Prenner et al., J. Biol. Chem. 299, 105038-10554 (2023)]. Although protein nitration is assumed to proceed via nonspecific chemical mechanisms, it is highly selective, suggesting the possibility of enzymatic catalysis. Here, we showed that glyoxalase domain-containing protein 4 (GLOD4), a previously uncharacterized protein

Enzymatic synthesis of fatty acid esters of trehalose: Process optimization, characterization of the esters and evaluation of their bioactivities.

One-pot synthesis of caprylic, lauric and palmitic acid mono- and diesters of trehalose was catalyzed by the lipase Fermase CALB™ 10000. An optimized molar conversion of 35% of trehalose to its palmitate esters was obtained in acetone at 60 °C with a trehalose:palmitic acid molar ratio of 1:5 in 4 h. Trehalose fatty acid esters (THFAE) were purified by column chromatography and characterized using TLC, HPTLC, HR-MS, ATR-FTIR, and differential scanning calorimetry. THFAE were studied for their antimicrobial potential against four bacterial, and two fungal species. Trehalose monolaurate and treh

Solution conformations of Zika NS2B-NS3pro and its inhibition by natural products from edible plants.

The recent Zika viral (ZIKV) epidemic has been associated with severe neurological pathologies such as neonatal microcephaly and Guillain-Barre syndrome but unfortunately no vaccine or medication is effectively available yet. Zika NS2B-NS3pro is essential for the proteolysis of the viral polyprotein and thereby viral replication. Thus NS2B-NS3pro represents an attractive target for anti-Zika drug discovery/design. Here, we have characterized the solution conformations and catalytic parameters of both linked and unlinked Zika NS2B-NS3pro complexes and found that the unlinked complex manifested

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99

Characterization of a novel CYP19A1 (aromatase) R192H mutation causing virilization of a 46,XX newborn, undervirilization of the 46,XY brother, but no virilization of the mother during pregnancies.

BACKGROUND: P450 aromatase (CYP19A1) is essential for the biosynthesis of estrogens from androgen precursors. Mutations in the coding region of CYP19A1 lead to autosomal recessive aromatase deficiency. To date over 20 subjects have been reported with aromatase deficiency which may manifest during fetal life with maternal virilization and virilization of the external genitalia of a female fetus due to low aromatase activity in the steroid metabolizing fetal-placental unit and thus high androgen levels. During infancy, girls often have ovarian cysts and thereafter fail to enter puberty showing s

Eicosanoid signaling and vascular dysfunction: methylmercury-induced phospholipase D activation in vascular endothelial cells.

Mercury, especially methylmercury (MeHg), is implicated in the etiology of cardiovascular diseases. Earlier, we have reported that MeHg induces phospholipase D (PLD) activation through oxidative stress and thiol-redox alteration. Hence, we investigated the mechanism of the MeHg-induced PLD activation through the upstream regulation by phospholipase A2 (PLA2) and lipid oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) in the bovine pulmonary artery endothelial cells (BPAECs). Our results showed that MeHg significantly activated both PLA2 (release of [(3)H]arachidonic acid, AA) and

Structural basis of O-methylation of (2-heptyl-)1-hydroxyquinolin-4(1H)-one and related compounds by the heterocyclic toxin methyltransferase Rv0560c of Mycobacterium tuberculosis.

The S-adenosyl-L-methionine-dependent methyltransferase Rv0560c of Mycobacterium tuberculosis belongs to an orthologous group of heterocyclic toxin methyltransferases (Htm) which likely contribute to resistance of mycobacteria towards antimicrobial natural compounds as well as drugs. HtmM.t. catalyzes the methylation of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one (also known as 2-heptyl-4-hydroxyquinoline N-oxide), a potent inhibitor of respiratory electron transfer, its 1-hydroxyquinolin-4(1H)-one core (QNO), structurally related (iso)quinolones, and some mycobacteri

Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.

Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins 1 . Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD+ to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins2, 3. However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic

Expression, characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin.

A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was close

Structural basis for activation of an integral membrane protease by lipopolysaccharide.

Omptins constitute a unique family of outer membrane proteases that are widespread in Enterobacteriaceae. The plasminogen activator (Pla) of Yersinia pestis is an omptin family member that is very important for development of both bubonic and pneumonic plague. The physiological function of Pla is to cleave (activate) human plasminogen to form the plasma protease plasmin. Uniquely, lipopolysaccharide (LPS) is essential for the catalytic activity of all omptins, including Pla. Why omptins require LPS for enzymatic activity is unknown. Here, we report the co-crystal structure of LPS-free Pla in c

New Insights and Predictions into Complex Homogeneous Reactions Enabled by Computational Chemistry in Synergy with Experiments: Isotopes and Mechanisms.

Homogeneous catalysis and biocatalysis have been widely applied in synthetic, medicinal, and energy chemistry as well as synthetic biology. Driven by developments of new computational chemistry methods and better computer hardware, computational chemistry has become an essentially indispensable mechanistic "instrument" to help understand structures and decipher reaction mechanisms in catalysis. In addition, synergy between computational and experimental chemistry deepens our mechanistic understanding, which further promotes the rational design of new catalysts. In this Account, we summarize ne…