Targeting MarA N-terminal domain dynamics to prevent DNA binding.

Efflux is one of the mechanisms employed by Gram-negative bacteria to become resistant to routinely used antibiotics. The inhibition of efflux by targeting their regulators is a promising strategy to re-sensitize bacterial pathogens to antibiotics. AcrAB-TolC is the main resistance-nodulation-division efflux pump in Enterobacteriaceae.

Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency.

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation.

The role of bacterial transport systems in the removal of host antimicrobial peptides in Gram-negative bacteria.

Antibiotic resistance is a global issue that threatens our progress in healthcare and life expectancy. In recent years, antimicrobial peptides (AMPs) have been considered as promising alternatives to the classic antibiotics. AMPs are potentially superior due to their lower rate of resistance development, since they primarily target the bacterial membrane ('Achilles' heel' of the bacteria).

Structural Plasticity of LL-37 Indicates Elaborate Functional Adaptation Mechanisms to Bacterial Target Structures.

The human cathelicidin LL-37 is a multifunctional peptide of the human innate immune system. Among the various functions of LL-37, its antimicrobial activity is important in controlling the microorganisms of the human body. The target molecules of LL-37 in bacteria include membrane lipids, lipopolysaccharides (LPS), lipoteichoic acid (LTA), proteins, DNA and RNA.

Auxiliary active site mutations enhance the glycosynthase activity of a GH18 chitinase for polymerization of chitooligosaccharides.

Depolymerization of chitin results in chitooligosaccharides (COS) that induce immunostimulatory effects and disease protective responses and have many potential applications in agriculture and medicine. Isolation of bioactive COS with degree of polymerization (DP) larger than six from chitin hydrolyzates is hampered by their water insolubility. Enzymatic synthesis by exploiting the transglycosylation activity of GH18 chitinases offers a potential strategy to access oligomers in the range of bioactive DPs.

The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics.

The human cathelicidin LL-37 serves a critical role in the innate immune system defending bacterial infections. LL-37 can interact with molecules of the cell wall and perforate cytoplasmic membranes resulting in bacterial cell death. To test the interactions of LL-37 and bacterial cell wall components we crystallized LL-37 in the presence of detergents and obtained the structure of a narrow tetrameric channel with a strongly charged core.

Metal Positions and Translocation Pathways of the Dodecameric Ferritin-like Protein Dps.

Iron storage in biology is carried out by cage-shaped proteins of the ferritin superfamily, one of which is the dodecameric protein Dps. In Dps, four distinct steps lead to the formation of metal nanoparticles: attraction of ion-aquo complexes to the protein matrix, passage of these complexes through translocation pores, oxidation of these complexes at ferroxidase centers, and, ultimately, nanoparticle formation. In this study, we investigated Dps from to structurally characterize these steps for Co, Zn, and La ions.

Structure and function of the Ts2631 endolysin of Thermus scotoductus phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding.

To escape from hosts after completing their life cycle, bacteriophages often use endolysins, which degrade bacterial peptidoglycan. While mesophilic phages have been extensively studied, their thermophilic counterparts are not well characterized. Here, we present a detailed analysis of the structure and function of Ts2631 endolysin from thermophilic phage vB_Tsc2631, which is a zinc-dependent amidase.

Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity.

Chitin deacetylases (CDAs) act on chitin polymers and low molecular weight oligomers producing chitosans and chitosan oligosaccharides. Structurally-defined, partially deacetylated chitooligosaccharides produced by enzymatic methods are of current interest as bioactive molecules for a variety of applications. Among Pochonia chlamydosporia (Pc) annotated CDAs, gene pc_2566 was predicted to encode for an extracellular CE4 deacetylase with two CBM18 chitin binding modules.

The Human Antimicrobial Peptides Dermcidin and LL-37 Show Novel Distinct Pathways in Membrane Interactions.

Mammals protect themselves from inflammation triggered by microorganisms through secretion of antimicrobial peptides (AMPs). One mechanism by which AMPs kill bacterial cells is perforating their membranes. Membrane interactions and pore formation were investigated for α-helical AMPs leading to the formulation of three basic mechanistic models: the barrel stave, toroidal, and carpet model.

Structural remodeling and oligomerization of human cathelicidin on membranes suggest fibril-like structures as active species.

Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces.

Structural Snapshots and Loop Dynamics along the Catalytic Cycle of Glycosyltransferase GpgS.

Glycosyltransferases (GTs) play a central role in nature. They catalyze the transfer of a sugar moiety to a broad range of acceptor substrates. GTs are highly selective enzymes, allowing the recognition of subtle structural differences in the sequences and stereochemistry of their sugar and acceptor substrates.

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of .

The gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in have remained enigmatic.

Structural basis of phosphatidyl-myo-inositol mannosides biosynthesis in mycobacteria.

Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids of unique chemical structure found in the inner and outer membranes of the cell envelope of all Mycobacterium species. The PIM family of glycolipids comprises phosphatidyl-myo-inositol mono-, di-, tri-, tetra-, penta-, and hexamannosides with different degrees of acylation. PIMs are considered not only essential structural components of the cell envelope but also the precursors of lipomannan and lipoarabinomannan, two major lipoglycans implicated in host-pathogen interactions.

Understanding N-Acetyl-L-Glutamate Synthase Deficiency: Mutational Spectrum, Impact of Clinical Mutations on Enzyme Functionality, and Structural Considerations.

N-acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification of NAGS gene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. Overall, 36 NAGS mutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele.

Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA.

The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2.

A Native Ternary Complex Trapped in a Crystal Reveals the Catalytic Mechanism of a Retaining Glycosyltransferase.

Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes, including cell signaling, cell development, and host-pathogen interactions. Glycosyl transfer can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge.

Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence.

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence.

Functional dissection of N-acetylglutamate synthase (ArgA) of Pseudomonas aeruginosa and restoration of its ancestral N-acetylglutamate kinase activity.

In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation.

Mechanism of arginine regulation of acetylglutamate synthase, the first enzyme of arginine synthesis.

N-acetyl-L-glutamate synthase (NAGS), the first enzyme of arginine biosynthesis in bacteria/plants and an essential urea cycle activator in animals, is, respectively, arginine-inhibited and activated. Arginine binds to the hexameric ring-forming amino acid kinase (AAK) domain of NAGS. We show that arginine inhibits Pseudomonas aeruginosa NAGS by altering the functions of the distant, substrate binding/catalytic GCN5-related N-acetyltransferase (GNAT) domain, increasing K(m)(Glu), decreasing V(max) and triggering substrate inhibition by AcCoA.

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor.

N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain.