Oximic compounds as potential inhibitors of metacaspase-2 (TbMCA2) of Trypanosoma brucei.

Metacaspases are a distinct class of cysteine proteases predominantly found in plants, fungi, and protozoa, crucial for regulating programmed cell death (PCD). They possess unique structural features and differ markedly from caspases in their activation mechanisms and substrate specificities, with a notable preference for binding basic residues in substrates. In this study, we introduced vanillin-derived oximic compounds to explore their pharmaceutical potential.

Metacaspase of Saccharomyces cerevisiae (ScMCA-Ia) presents different catalytic cysteine in a processed and non-processed form.

Metacaspases are cysteine proteases belonging to the CD clan of the C14 family. They possess important characteristics, such as specificity for cleavage after basic residues (Arg/Lys) and dependence on calcium ions to exert their catalytic activity. They are defined by the presence of a large subunit (p20) and a small subunit (p10) and are classified into types I, II, and III.

Genetic and biochemical characterization of BIM-1, a novel acquired subgroup B1 MBL found in a Pseudomonas sp. strain from the Brazilian Amazon region.

Objectives: To characterize a novel acquired MBL, BIM-1, in a Pseudomonas #2 (subgroup P. guariconensis) strain isolated from the Aurá river located in the Brazilian Amazon hydrographic basin.

Methods: WGS using an Illumina® MiSeq System was used to characterize the genome of Pseudomonas sp.

Kinetics Analysis of β-Lactams Hydrolysis by OXA-50 Variants of .

is an opportunist pathogen usually associated with life threatening infections and exhibits a set of intrinsic and acquired antimicrobial mechanisms. Although resistance to penicillins-like compounds is commonly associated with the chromosomal -derived cephalosporinases β-lactamase, the real contribution of OXA-50, a second chromosomally encoded β-lactamase, remains unclear. In this study, we characterized the biochemical properties of OXA-50, OXA-488, and OXA-494.

Probing the Conformational States of Thimet Oligopeptidase in Solution.

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used.

Enzymatic diversity of filamentous fungi isolated from forest soil incremented by sugar cane solid waste.

Fungi are natural degraders of organic matter which can produce enzymes for many industrial and biotechnological applications. In this context, crude enzymatic extracts of fungal isolates were evaluated regarding their hydrolytic and ligninolytic abilities. The fungal strains were isolated from soil samples from Atlantic Rain Forest Park incremented with sugar cane biomass (filter cake), which allowed the selection of efficient lignocellulolytic enzymes.

CPP-Ala-Ala-Tyr-PABA inhibitor analogs with improved selectivity for neurolysin or thimet oligopeptidase.

Thimet oligopeptidase (TOP, EC 3.4.24.

Genetic and biochemical characterization of GES-16, a new GES-type β-lactamase with carbapenemase activity in Serratia marcescens.

We evaluated the genetic environment of bla found in 2 carbapenem-resistant Serratia marcescens clinical isolates recovered from patients hospitalized at a tertiary hospital located in Rio de Janeiro, Brazil. We also compared the kinetics constants for GES-16 and GES-5 against several β-lactams. Both S.

On the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectors.

Biosynthetic incorporation of non-canonic amino acids is an attractive strategy to introduce new properties in recombinant proteins. Trp analogs can be incorporated in recombinant proteins replacing regular Trp during protein translation into a Trp-auxotrophic cell host. This straightforward method however, is limited to few analogs recognized and accepted by the cellular protein production machinery.

Processing of metacaspase 2 from Trypanosoma brucei (TbMCA2) broadens its substrate specificity.

Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P. In contrast to caspases, they do not require processing or dimerization for activity.

Development of Chemically Defined Media to Express Trp-Analog-Labeled Proteins in a Lactococcus lactis Trp Auxotroph.

Chemically defined media for growth of Lactococcus lactis strains contain about 50 components, making them laborious and expensive growth media. However, they are crucial for metabolism studies as well as for expression of heterologous proteins labeled with unnatural amino acids. In particular, the L.

Intermediate Tyrosyl Radical and Amyloid Structure in Peroxide-Activated Cytoglobin.

We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping.

Characterization of BKC-1 class A carbapenemase from Klebsiella pneumoniae clinical isolates in Brazil.

Three Klebsiella pneumoniae clinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all β-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for all K.

Characterization of angiotensin I-converting enzyme from anterior gills of the mangrove crab Ucides cordatus.

Angiotensin I-converting enzyme (ACE) is a well-known metallopeptidase that is found in vertebrates, invertebrates and bacteria. We isolated from the anterior gill of the crab Ucides cordatus an isoform of ACE, here named crab-ACE, which presented catalytic properties closely resembling to those of mammalian ACE. The enzyme was purified on Sepharose-lisinopril affinity chromatography to apparent homogeneity and a band of about 72 kDa could be visualized after silver staining and Western blotting.

Recycling of the high valence States of heme proteins by cysteine residues of THIMET-oligopeptidase.

The peptidolytic enzyme THIMET-oligopeptidase (TOP) is able to act as a reducing agent in the peroxidase cycle of myoglobin (Mb) and horseradish peroxidase (HRP). The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme. EPR (electron paramagnetic resonance) analysis using DBNBS spin trapping revealed that TOP also prevented the formation of tryptophanyl radical in Mb challenged by H2O2.

Substrate specificity and the effect of calcium on Trypanosoma brucei metacaspase 2.

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca(2+) on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P1 position of peptide substrates and that this is most probably a result of hydrogen bonding from the P1 residue to Asp95 and Asp211 in TbMCA2.

Hysteretic behavior of proprotein convertase 1/3 (PC1/3).

The proprotein convertases (PCs) are calcium-dependent proteases responsible for processing precursor proteins into their active forms in eukariotes. The PC1/3 is a pivotal enzyme of this family that participates in the proteolytic maturation of prohormones and neuropeptides inside the regulated secretory pathway. In this paper we demonstrate that mouse proprotein convertase 1/3 (mPC1/3) has a lag phase of activation by substrates that can be interpreted as a hysteretic behavior of the enzyme for their hydrolysis.

Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr(607) residue.

Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 ((600)SHIFAGGYAAGYYSY(614)).

Catalytic properties of thimet oligopeptidase H600A mutant.

Thimet oligopeptidase (EC 3.4.24.

Mitochondrial intermediate peptidase: expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates.

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column.

Plasma and dialysate IL-6 and VEGF concentrations are associated with high peritoneal solute transport rate.

Background: It has been speculated that increased levels of circulating or intraperitoneal pro-inflammatory cytokines such as interleukin 6, and pro-angiogenic vascular endothelial growth factor (VEGF) may contribute to high peritoneal small-solute transport rate (PSTR) in continuous ambulatory peritoneal dialysis (CAPD) patients. In this study we evaluated possible relationships between plasma and dialysate IL-6 and VEGF levels and PSTR.

Methods: Forty CAPD patients (mean age+/-SD of 58+/-14 years) with no apparent inflammation process or disease, who had been on CAPD for 19+/-15 months (range 3-56 months) were included in the study.