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Crystal structure of a glutamate-1-semialdehyde-aminomutase from Pseudomonas aeruginosa PAO1.

The C5 pathway in bacteria is responsible for the synthesis of 5-aminolevulinic acid, which forms the core skeleton of cofactors required for metabolism. One of the key actors in this pathway is a pyridoxamine-5'-phosphate (PMP)/pyridoxal-5'-phosphate (PLP) dependent enzyme called glutamate-1-semialdehyde aminomutase (GSAM). In this study, we crystallized the expression product of the uncharacterized pa4088 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The resulting high-resolution structure confirms it to be a member of the GSAM family. Continuous electron density indicates the presence of a PLP cofactor with a Schiff base linkage between the PLP cofactor and the ε-amino group of Lys286. A crystal structure of a K286A mutant in complex with PMP is also reported. As GSAM enzymes are not present in mammalian cells, this work provides a starting point for the investigation of GSAM as a target for drug development against P. aeruginosa infection.

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Biochemical and biophysical studies of Helicobacter pylori arginine decarboxylase, an enzyme important for acid adaptation in host.

Despite importance of arginine decarboxylase (ADC: EC 4.1.1.19) of Helicobacter pylori (H. pylori) 26695 pathogenic strain for acid adaptation in host, the enzyme has not yet been studied at a molecular level. Using combined approaches that include kinetic assays, site-directed mutagenesis, circular dichroism, heat-induced denaturation, analytical gel-filtration, and homology modeling, we report here a detailed investigation of H. pylori ADC. The pyridoxal 5'-phosphate (PLP)-dependent enzyme exhibits higher catalytic activity in the presence of Mg ions at pH ∼8.5. Unlike other bacterial ADCs, this homolog exists as a hexamer. The higher thermal stability (T ∼65.8 ± 0.2 °C) of the enzyme observed from the heat-induced circular dichroism measurements indicates its secondary structural stabilization in the presence of PLP. The kinetic parameters K and k of the enzyme are determined to be 3.4 ± 0.2 mM and 55.2 ± 1.0 min , respectively. We elucidate that Cys487, a conserved residue located at the active-site, is involved in the catalysis, whose pK value was estimated to be ∼7.2. The homology model of the protein show conserved α/β TIM barrel and β-sandwich domains, which are characteristic features of fold III decarboxylases. A lower sequence identity (∼21%) of this enzyme compared with its human counterpart has enabled us to screen putative inhibitors of H. pylori ADC. We found that α-difluoromethylarginine inhibits the activity of the H. pylori enzyme competitively with a K value ∼118 µM and thus it can serve as a basis to design inhibitors with higher efficacy against this ADC. © 2018 IUBMB Life, 2018.

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STRATEGIES TO ENHANCE RECRUITMENT METHODS IN PHANTOM LIMB PAIN CLINICAL TRIALS.

Phantom Limp Pain (PLP) was first described in 1551. To date, its mechanisms and novel interventions remain mostly untested. Only limited conclusions can be drawn from few and small randomized clinical trials (RCTs) on PLP. In this scenario, recruitment strategies are crucial in order to overcome inherent challenges to recruit PLP subjects for clinical trials. Although there are many methods to enhance recruitment and also retention, in this article we discuss these methods based on a common topic: dissemination. We summarize and discuss 10 strategies of recruitment related to the dissemination of information based on the notion that an increase in trial awareness may lead to both increased recruitment and also increased external generalizability. In addition, in our discussion we included insights based on our experience recruiting PLP patients for our large NIH-sponsored clinical trial. Although specific regulatory considerations need to be considered when choosing the methods of recruitment, which may vary across different countries and Institutional Review Boards (IRBs), these strategies may be applicable to most of research settings.

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Cyclization of PLP peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis.

We report the novel synthesis of cyclic PLP (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H replaces W and F-K replace H as TCR contacts, which may explain the difference on each peptide's response.

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Relationship between plasma levels of homocysteine and the related B vitamins in patients with hemodialysis adequacy or inadequacy.

Hemodialysis (HD) with dialysis adequacy could increase the excretion of B vitamins (folate, vitamin B, and B) and raise the plasma level of homocysteine. Here we determined the associations of plasma homocysteine with B vitamins in patients with HD adequacy or inadequacy.

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Structural and functional studies of Spr1654: an essential aminotransferase in teichoic acid biosynthesis in .

Spr1654 from plays a key role in the production of unusual sugars, presumably functioning as a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase. Spr1654 was predicted to catalyse the transferring of amino group to form the amino sugar 2-acetamido-4-amino-2, 4, 6-trideoxygalactose moiety (AATGal), representing a crucial step in biosynthesis of teichoic acids in We have determined the crystal structures of the apo-, PLP- and PMP-bound forms of Spr1654. Spr1654 forms a homodimer, in which each monomer contains one active site. Using spectrophotometry and based on absorbance profiles of PLP- and PMP-formed enzymes, our results indicate that l-glutamate is most likely the preferred amino donor. Structural superposition of the crystal structures of Spr1654 on previously determined structures of other sugar aminotransferases in complex with glutamate and/or UDP-activated sugar allowed us to identify key Spr1654 residues for ligand binding and catalysis. The crystal structures of Spr1654 and in complex with PLP and PMP can direct the future rational design of novel therapeutic compounds against .

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Genotypic and Lipid Analyses of Strains From the Archaeal Genus Reveal Insights Into Their Taxonomy, Divergence, and Population Structure.

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge.

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Crystal Structures of Cystathionine β-Synthase from Saccharomyces cerevisiae: One Enzymatic Step at a Time.

Cystathionine β-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of HS biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.

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Mini-Review: Ergothioneine and Ovothiol Biosyntheses, an Unprecedented Trans-Sulfur Strategy in Natural Product Biosynthesis.

As one of the most abundant elements on earth, sulfur is part of many small molecular metabolites and is key to their biological activities. Over the past few decades, some general strategies have been discovered for the incorporation of sulfur into natural products. In this review, we summarize recent efforts in elucidating the biosynthetic details for two sulfur-containing metabolites, ergothioneine and ovothiol. Their biosyntheses involve an unprecedented trans-sulfur strategy, a combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation reaction and a PLP enzyme-mediated C-S lyase reaction.

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Clemastine effects in rat models of a myelination disorder.

Pelizaeus Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system caused by impaired differentiation of oligodendrocytes. This study was prompted by findings that anti-muscarinic compounds enhance oligodendrocyte differentiation and remyelination in vitro. One of these compounds, clemastine fumarate, is licensed for treatment of allergic conditions. We tested whether clemastine fumarate can promote myelination in two rodent PMD-models, the myelin deficient and the PLP transgenic rat.

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