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1.
ACS Synth Biol ; 11(9): 3078-3087, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36065523

RESUMO

Mersacidin is an antimicrobial class II lanthipeptide. Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by intramolecular lanthionine rings. These rings give lanthipeptides their bioactive structure and stability. RiPPs are produced from a gene cluster that encodes a precursor peptide and its dedicated unique modification enzymes. The field of RiPP engineering aims to recombine modification enzymes from different RiPPs to modify new substrates, resulting in new-to-nature molecules with novel or improved functionality. The enzyme MrsM from the mersacidin gene cluster installs the four lanthionine rings of mersacidin, including the uniquely small ring A. By applying MrsM in RiPP engineering, this ring could be installed in linear peptides to achieve stabilization by a very small lanthionine or to create small lanthionine-stabilized modules for chemical modification. However, the formation of unique intramolecular structures like that of mersacidin's ring A can be very stringent. Here, the formation of ring A of mersacidin is characterized by mutagenesis. A range of truncated mersacidin variants was made to identify the smallest possible construct in which this ring could still be formed. Additionally, mutants were created to study the flexibility of ring A formation. It was found that although the formation of ring A is stringent, it can be formed in a core peptide as small as five amino acids. The truncated mersacidin core peptide CTFAL is the smallest ribosomally produced lanthipeptide reported to date, and it has exciting prospects as a new module for application in RiPP engineering.


Assuntos
Bacteriocinas , Alanina/análogos & derivados , Alanina/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Peptídeos/metabolismo , Sulfetos
2.
Pestic Biochem Physiol ; 187: 105194, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36127066

RESUMO

Chlorantraniliprole (CAP), a representative bisamide insecticide, is widely used in rice fields around the world, posing potential toxicity risks to aquatic organisms. In this study, we examined the effects of exposure to CAP on growth and metabolic phenotype of zebrafish (Danio rerio) and oxidative stress and apoptosis in the liver of zebrafish (Danio rerio). First, we identified that CAP had a low bioaccumulation in zebrafish. Subsequently, growth phenotype analysis revealed that CAP could significantly increase liver weight and liver index in zebrafish. In addition, we found that CAP exposure could cause significant changes in indicators of oxidative stress, resulting in a significant increase in the content of malondialdehyde (MDA), causing oxidative stress in the liver of zebrafish. Meanwhile, the expression levels of apoptosis-related genes were also significantly changed and apoptosis was promoted in the liver of zebrafish with CAP exposure. Importantly, the results of metabolomics analysis shown that CAP exposure could significantly disrupt the metabolic phenotype of zebrafish, interfering with multiple metabolic pathways, mainly including valine, leucine and isoleucine biosynthesis and degradation, alanine, aspartate and glutamate metabolism and d-glutamine and D-glutamate metabolism. Last but not least, correlation analysis identified strong links between changes in liver function involving oxidative stress and apoptosis and changes in metabolic phenotype of zebrafish following CAP exposure. In brief, these results indicate that potential environmental risks of CAP to aquatic organisms should receive more attention.


Assuntos
Inseticidas , Poluentes Químicos da Água , Alanina/metabolismo , Animais , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Ácido Glutâmico , Glutamina/genética , Glutamina/metabolismo , Glutamina/farmacologia , Inseticidas/farmacologia , Isoleucina/genética , Isoleucina/metabolismo , Isoleucina/farmacologia , Leucina , Fígado , Malondialdeído/metabolismo , Fenótipo , Valina/farmacologia , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , ortoaminobenzoatos
3.
J Am Chem Soc ; 144(37): 17022-17032, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36084022

RESUMO

NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.


Assuntos
Hidrogenase , Alanina/metabolismo , Ácido Aspártico/metabolismo , Domínio Catalítico , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Hidrogenase/química , Hydrogenophilaceae , Ferro/química , Ligantes , NAD/metabolismo , Níquel/química , Oxirredução , Oxigênio/química
4.
Sci Signal ; 15(752): eabn8359, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36126115

RESUMO

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.


Assuntos
HIV-1 , Domínios de Homologia de src , Alanina/metabolismo , HIV-1/metabolismo , Humanos , Tirosina/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
5.
Int J Mol Sci ; 23(15)2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35955589

RESUMO

Sirex noctilio Fabricius (Hymenoptera Siricidae) is a major quarantine pest responsible for substantial economic losses in the pine industry. To achieve better pest control, (Z)-3-decen-ol was identified as the male pheromone and used as a field chemical trapping agent. However, the interactions between odorant-binding proteins (OBPs) and pheromones are poorly described. In this study, SnocOBP9 had a higher binding affinity with Z3D (Ki = 1.53 ± 0.09 µM) than other chemical ligands. Molecular dynamics simulation and binding mode analysis revealed that several nonpolar residues were the main drivers for hydrophobic interactions between SnocOBP9 and Z3D. Additionally, computational alanine scanning results indicated that five amino acids (MET54, PHE57, PHE71, PHE74, LEU116) in SnocOBP9 could potentially alter the binding affinity to Z3D. Finally, we used single-site-directed mutagenesis to substitute these five residues with alanine. These results imply that the five residues play crucial roles in the SnocOBP9-Z3D complex. Our research confirmed the function of SnocOBP9, uncovered the key residues involved in SnocOBP9-Z3D interactions, and provides an inspiration to improve the effects of pheromone agent traps.


Assuntos
Himenópteros , Receptores Odorantes , Alanina/metabolismo , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Feromônios/metabolismo , Ligação Proteica , Receptores Odorantes/metabolismo
6.
Int J Mol Sci ; 23(15)2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35955719

RESUMO

Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.


Assuntos
Proteínas de Transporte , Ceramidas , Alanina/metabolismo , Aminoácidos Básicos/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Complexo de Golgi/metabolismo , Humanos , Fosfatidilinositóis/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Serina/metabolismo
7.
Magn Reson Imaging ; 93: 189-194, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36029935

RESUMO

The potential use of alanine as an MRI contrast agent was investigated. The relaxation properties of alanine solutions were measured at 9.4 T. The T2 relaxivity caused by the chemical exchange (R2ex) between amine protons and water protons was 0.10 mM-1 s-1 at 37 °C. As a demonstration, alanine uptake in a mouse xenograft model of U-87 MG glioblastoma was measured using MRI, and was compared with immunohistochemistry staining of ASCT2, a transporter that imports amino acids into cancer cells. Statistically significant (p = 0.0079) differences in ASCT2 distribution were found between regions that show strong and weak alanine uptake in MRI. To better understand the influence of perfusion, the effect of ASCT2 inhibition on the alanine uptake in MRI was investigated, and dynamic contrast enhanced MRI was compared with alanine MRI.


Assuntos
Sistema ASC de Transporte de Aminoácidos , Glioblastoma , Alanina/metabolismo , Sistema ASC de Transporte de Aminoácidos/química , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Glioblastoma/diagnóstico por imagem , Xenoenxertos , Humanos , Imageamento por Ressonância Magnética , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Prótons
8.
mBio ; 13(4): e0171422, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35880880

RESUMO

Human immunodeficiency virus type-1 (HIV-1) infection is potently inhibited by human myxovirus resistance 2 (MX2/MxB), which binds to the viral capsid and blocks the nuclear import of viral DNA. We have recently shown that phosphorylation is a key regulator of MX2 antiviral activity, with phosphorylation of serine residues at positions 14, 17, and 18 repressing MX2 function. Here, we extend the study of MX2 posttranslational modifications and identify serine and threonine phosphorylation in all domains of MX2. By substituting these residues with aspartic acid or alanine, hence mimicking the presence or absence of a phosphate group, respectively, we identified key positions that control MX2 antiviral activity. Aspartic acid substitutions of residues Ser306 or Thr334 and alanine substitutions of Thr343 yielded proteins with substantially reduced antiviral activity, whereas the presence of aspartic acid at positions Ser28, Thr151, or Thr343 resulted in enhanced activity: referred to as hypermorphic mutants. In some cases, these hypermorphic mutations, particularly when paired with other MX2 mutations (e.g., S28D/T151D or T151D/T343A) acquired the capacity to inhibit HIV-1 capsid mutants known to be insensitive to wild-type MX2, such as P90A or T210K, as well as MX2-resistant retroviruses such as equine infectious anemia virus (EIAV) and murine leukemia virus (MLV). This work highlights the complexity and importance of MX2 phosphorylation in the regulation of antiviral activity and in the selection of susceptible viral substrates. IMPORTANCE Productive infection by human immunodeficiency virus type-1 (HIV-1) requires the import of viral replication complexes into the nuclei of infected cells. Myxovirus resistance 2 (MX2/MxB) blocks this step, halting nuclear accumulation of viral DNA and virus replication. We recently demonstrated how phosphorylation of a stretch of three serines in the amino-terminal domain of MX2 inhibits the antiviral activity. Here, we identify additional positions in MX2 whose phosphorylation status reduces or enhances antiviral function (hypomorphic and hypermorphic variants, respectively). Importantly, hypermorphic mutant proteins not only increased inhibitory activity against wild-type HIV-1 but can also exhibit antiviral capabilities against HIV-1 capsid mutant viruses that are resistant to wild-type MX2. Furthermore, some of these proteins were also able to inhibit retroviruses that are insensitive to MX2. Therefore, we propose that phosphorylation comprises a major element of MX2 regulation and substrate determination.


Assuntos
Infecções por HIV , HIV-1 , Alanina/metabolismo , Animais , Antivirais/metabolismo , Ácido Aspártico/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , DNA Viral/metabolismo , HIV-1/fisiologia , Cavalos/genética , Humanos , Camundongos , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Fosforilação , Serina , Replicação Viral
9.
Molecules ; 27(14)2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35889320

RESUMO

The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphorylation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to the presence of ionizable negatively-charged groups. To circumvent these limitations, several prodrug approaches have been proposed. Herein, we hypothesized that the conjugation or combination of the lipophilic amide bond with nucleotide-based tenofovir (TFV) (1) could improve the anti-HIV activity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated with the amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty ester hydrocarbon chains to synthesize 43 derivatives. Several classes of derivatives were synthesized. The synthesized compounds were characterized by 1H NMR, IR, UV, and mass spectrometry. In addition, several of the synthesized compounds were evaluated as racemic mixtures for anti-HIV activity in vitro in a single round infection assay using TZM-bl cells at 100 ng/mL. TFV (1) was used as a positive control and inhibited HIV infection by 35%. Among all the evaluated compounds, the disubstituted heptanolyl ester alanine phosphonamidate with naphthol oleate (69), pentanolyl ester alanine phosphonamidate with phenol oleate (62), and butanolyl ester alanine phosphonamidate with naphthol oleate (87) ester conjugates of TFV were more potent than parent drug TFV with 79.0%, 76.5%, 71.5% inhibition, respectively, at 100 ng/mL. Furthermore, two fatty acyl amide conjugates of tenofovir alafenamide (TAF) were synthesized and evaluated for comparative studies with TAF and TFV conjugates. Tetradecanoyl TAF conjugate 95 inhibited HIV infection by 99.6% at 100 ng/mL and showed comparable activity to TAF (97-99% inhibition) at 10-100 ng/mL but was more potent than TAF when compared at molar concentration.


Assuntos
Fármacos Anti-HIV , Infecções por HIV , HIV-1 , Alanina/metabolismo , Amidas/metabolismo , Fármacos Anti-HIV/uso terapêutico , Ésteres/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Humanos , Naftóis/metabolismo , Nucleotídeos/metabolismo , Ácido Oleico/metabolismo , Tenofovir/farmacologia
10.
Microb Cell Fact ; 21(1): 146, 2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35843946

RESUMO

BACKGROUND: Cyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce. RESULTS: We isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production. CONCLUSIONS: In this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.


Assuntos
Aspergillus oryzae , Alanina/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Dipeptídeos/metabolismo , Triptofano/metabolismo
11.
J Mol Neurosci ; 72(9): 1965-1976, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35852782

RESUMO

Altered amine transporter function, phosphorylation, and association with interacting proteins are evident in animals with a history of psychostimulant exposure. Our previous studies have shown that the Thr258/Ser259 motif in the norepinephrine transporter (NET) is involved in amphetamine (AMPH)-mediated NET regulation and behavior. However, the neurobiological consequences of in vivo Thr258/Ser259-dependent NET regulation in an intact animal model are unclear. Therefore, we generated a viable construct-valid NET-Thr258Ala/Ser259Ala (NET-T258A/S259A) mouse model using CRISPR/Cas9 technology by replacing Thr258/Ser259 motif with Ala258/Ala259 motif. NET-T258A/S259A mice have a birth rate consistent with Mendelian inheritance ratios. Both male and female homozygous NET-T258A/S259A mice are viable, display normal growth and general health, and exhibit normal body weight (sex-dependent) and total activity in the open field similar to their wild-type (WT) littermates. NET-T258A/S259A mice showed reduced NET function in the prefrontal cortex (PFC) compared to WT mice while NET function in the nucleus accumbens (NAc) remained unchanged. Compared to respective WT counterparts, NET-T258A/S259A males but not females showed significantly reduced locomotor activation in response to acute AMPH administration and significantly reduced AMPH-induced conditioned place preference (CPP). When tested in the males only, acute AMPH administration inhibited NET function and surface expression in the WT NAc but not in the NET-T258A/S259A NAc while AMPH administration inhibited DAT function and surface expression in the NAc of both WT and NET-T258A/S259A mice. Collectively, our findings reveal that the mice carrying the T258A/S259A mutation in NET gene display brain region-specific differences in NET functional expression and blunted response to AMPH.


Assuntos
Anfetamina , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Alanina/genética , Alanina/metabolismo , Anfetamina/farmacologia , Animais , Regulação para Baixo , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Mutação , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Serina , Treonina/genética , Treonina/metabolismo
12.
Theor Appl Genet ; 135(8): 2817-2831, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35779128

RESUMO

KEY MESSAGE: An alanine to valine mutation of glutamyl-tRNA reductase's 510th amino acid improves 5-aminolevulinic acid synthesis in rice. 5-aminolevulinic acid (ALA) is the common precursor of all tetrapyrroles and plays an important role in plant growth regulation. ALA is synthesized from glutamate, catalyzed by glutamyl-tRNA synthetase (GluRS), glutamyl-tRNA reductase (GluTR), and glutamate-1-semialdehyde aminotransferase (GSAT). In Arabidopsis, ALA synthesis is the rate-limiting step in tetrapyrrole production via GluTR post-translational regulations. In rice, mutations of GluTR and GSAT homologs are known to confer chlorophyll deficiency phenotypes; however, the enzymatic activity of rice GluRS, GluTR, and GSAT and the post-translational regulation of rice GluTR have not been investigated experimentally. We have demonstrated that a suppressor mutation in rice partially reverts the xantha trait. In the present study, we first determine that the suppressor mutation results from a G → A nucleotide substitution of OsGluTR (and an A → V change of its 510th amino acid). Protein homology modeling and molecular docking show that the OsGluTRA510V mutation increases its substrate binding. We then demonstrate that the OsGluTRA510V mutation increases ALA synthesis in Escherichia coli without affecting its interaction with OsFLU. We further explore homologous genes encoding GluTR across 193 plant species and find that the amino acid (A) is 100% conserved at the position, suggesting its critical role in GluTR. Thus, we demonstrate that the gain-of-function OsGluTRA510V mutation underlies suppression of the xantha trait, experimentally proves the enzymatic activity of rice GluRS, GluTR, and GSAT in ALA synthesis, and uncovers conservation of the alanine corresponding to the 510th amino acid of OsGluTR across plant species.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Oryza , Alanina/genética , Alanina/metabolismo , Aldeído Oxirredutases , Ácido Aminolevulínico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Simulação de Acoplamento Molecular , Mutação , Oryza/genética , Oryza/metabolismo , Valina/genética , Valina/metabolismo
13.
Microbiol Spectr ; 10(4): e0173422, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-35758683

RESUMO

Vancomycin and ß-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is d-alanine-d-alanine (d-Ala-d-Ala), whereas that of ß-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidase DacA on vancomycin and ß-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of DacA induced sensitivity to most ß-lactams, whereas it induced strong resistance toward vancomycin. Notably, both phenotypes did not have a strong association with ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by an increased amount of decoy d-Ala-d-Ala residues within PG, whereas ß-lactam sensitivity was associated with physical interactions between DacA and PBPs. The presence of an OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened ß-lactam sensitivity. Collectively, our results revealed two distinct functions of DacA, which involved inverse modulation of bacterial resistance to clinically important antibiotics, ß-lactams and vancomycin, and presented evidence for a link between DacA and PBPs. IMPORTANCE Bacterial PG hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. We have revealed two distinct functions of PG carboxypeptidase DacA with respect to antibiotic resistance. The deletion of DacA led to sensitivity to most ß-lactams, while it caused strong resistance to vancomycin. Our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of ß-lactam antibiotics.


Assuntos
Peptidoglicano , beta-Lactamas , Alanina/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Carboxipeptidases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Vancomicina/metabolismo , Vancomicina/farmacologia , Resistência a Vancomicina , beta-Lactamas/metabolismo , beta-Lactamas/farmacologia
14.
J Biol Chem ; 298(8): 102195, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35760102

RESUMO

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine-aldimine complex to a substrate-aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.


Assuntos
Bacteroidetes , Ácido Cisteico , Proteína de Transporte de Acila , Alanina/metabolismo , Bacteroidetes/metabolismo , Lipídeos , Fosfato de Piridoxal/metabolismo
15.
Chembiochem ; 23(17): e202200171, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-35695820

RESUMO

The carbon backbone of biotin is constructed from the C7 di-acid pimelate, which is converted to an acyl-CoA thioester by an ATP-dependent, pimeloyl-CoA synthetase (PCAS, encoded by BioW). The acyl-thioester is condensed with ʟ-alanine in a decarboxylative, Claisen-like reaction to form an aminoketone (8-amino-7-oxononanoic acid, AON). This step is catalysed by the pyridoxal 5'-phosphate (PLP)-dependent enzyme (AON synthase, AONS, encoded by BioF). Distinct versions of Bacillus subtilis BioW (BsBioW) and E. coli BioF (EcBioF) display strict substrate specificity. In contrast, a BioW-BioF fusion from Corynebacterium amycolatum (CaBioWF) accepts a wider range of mono- and di-fatty acids. Analysis of the active site of the BsBioW : pimeloyl-adenylate complex suggested a key role for a Phe (F192) residue in the CaBioW domain; a F192Y mutant restored the substrate specificity to pimelate. This surprising substrate flexibility also extends to the CaBioF domain, which accepts ʟ-alanine, ʟ-serine and glycine. Structural models of the CaBioWF fusion provide insight into how both domains interact with each other and suggest the presence of an intra-domain tunnel. The CaBioWF fusion catalyses conversion of various fatty acids and amino acids to a range of AON derivatives. Such unexpected, natural broad substrate scope suggests that the CaBioWF fusion is a versatile biocatalyst that can be used to prepare a number of aminoketone analogues.


Assuntos
Proteínas de Bactérias , Biotina , Coenzima A Ligases , Acil Coenzima A/metabolismo , Alanina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotina/biossíntese , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Fosfato de Piridoxal/metabolismo , Especificidade por Substrato
16.
Beijing Da Xue Xue Bao Yi Xue Ban ; 54(3): 532-540, 2022 Jun 18.
Artigo em Chinês | MEDLINE | ID: mdl-35701132

RESUMO

OBJECTIVE: To explore the amino acid metabolomics characteristics of myeloid-derived suppressor cells (MDSCs) in mice with sepsis induced by the cecal ligation and puncture (CLP). METHODS: The sepsis mouse model was prepared by CLP, and the mice were randomly divided into a sham operation group (sham group, n = 10) and a CLP model group (n = 10). On the 7th day after the operation, 5 mice were randomly selected from the surviving mice in each group, and the bone marrow MDSCs of the mice were isolated. Bone marrow MDSCs were separated to measure the oxygen consumption rate (OCR) by using Agilent Seahorse XF technology and to detect the contents of intracellular amino acids and oligopeptides through ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) technology. Different metabolites and potential biomarkers were analyzed by univariate statistical analysis and multivariate statistical analysis. The major metabolic pathways were enriched using the small molecular pathway database (SMPDB). RESULTS: The proportion of MDSCs in the bone marrow of CLP group mice (75.53% ± 6.02%) was significantly greater than that of the sham group (43.15%± 7.42%, t = 7.582, P < 0.001), and the basal respiratory rate [(50.03±1.20) pmol/min], maximum respiration rate [(78.07±2.57) pmol/min] and adenosine triphosphate (ATP) production [(25.30±1.21) pmol/min] of MDSCs in the bone marrow of CLP group mice were significantly greater than the basal respiration rate [(34.53±0.96) pmol/min, (t = 17.41, P < 0.001)], maximum respiration rate [(42.57±1.87) pmol/min, (t = 19.33, P < 0.001)], and ATP production [(12.63±0.96) pmol/min, (t = 14.18, P < 0.001)] of sham group. Leucine, threonine, glycine, etc. were potential biomarkers of septic MDSCs (all P < 0.05). The increased amino acids were mainly enriched in metabolic pathways, such as malate-aspartate shuttle, ammonia recovery, alanine metabolism, glutathione metabolism, phenylalanine and tyrosine metabolism, urea cycle, glycine and serine metabolism, ß-alanine metabolism, glutamate metabolism, arginine and proline metabolism. CONCLUSION: The enhanced mitochondrial oxidative phosphorylation, malate-aspartate shuttle and alanine metabolism in MDSCs of CLP mice may provide raw materials for mitochondrial aerobic respiration, thereby promoting the immunosuppressive function of MDSCs. Blocking the above metabolic pathways may reduce the risk of secondary infection in sepsis and improve the prognosis.


Assuntos
Células Supressoras Mieloides , Sepse , Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Animais , Ácido Aspártico/metabolismo , Biomarcadores/metabolismo , Cromatografia Líquida , Glicina/metabolismo , Malatos/metabolismo , Camundongos , Células Supressoras Mieloides/metabolismo , Sepse/complicações , Espectrometria de Massas em Tandem
17.
Biochemistry ; 61(13): 1298-1312, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35699437

RESUMO

Two plastoquinone electron acceptors, QA and QB, are present in Photosystem II (PS II) with their binding sites formed by the D2 and D1 proteins, respectively. A hexacoordinate non-heme iron is bound between QA and QB by D2 and D1, each providing two histidine ligands, and a bicarbonate that is stabilized via hydrogen bonds with D2-Tyr244 and D1-Tyr246. Both tyrosines and bicarbonate are conserved in oxygenic photosynthetic organisms but absent from the corresponding quinone-iron electron acceptor complex of anoxygenic photosynthetic bacteria. We investigated the role of D2-Tyr244 by introducing mutations in the cyanobacterium Synechocystis sp. PCC 6803. Alanine, histidine, and phenylalanine substitutions were introduced creating the Y244A, Y244H, and Y244F mutants. Electron transfer between QA and QB was impaired, the back-reaction with the S2 state of the oxygen-evolving complex was modified, and PS II assembly was disrupted, with the Y244A strain being more affected than the Y244F and Y244H mutants. The strains were also highly susceptible to photodamage in the presence of PS II-specific electron acceptors. Thermoluminescence and chlorophyll a fluorescence decay measurements indicated that the redox potential of the QA/QA- couple became more positive in the Y244F and Y244H mutants, consistent with bicarbonate binding being impacted. The replacement of Tyr244 by alanine also led to an insertion of two amino acid repeats from Gln239 to Ala249 within the DE loop of D2, resulting in an inactive PS II complex that lacked PS II-specific variable fluorescence. The 66 bp insertion giving rise to the inserted amino acids therefore created an obligate photoheterotrophic mutant.


Assuntos
Complexo de Proteína do Fotossistema II , Synechocystis , Alanina/metabolismo , Bicarbonatos/metabolismo , Clorofila/química , Clorofila A/metabolismo , Transporte de Elétrons , Histidina/genética , Histidina/metabolismo , Ferro/metabolismo , Complexo de Proteína do Fotossistema II/química , Quinonas/metabolismo , Synechocystis/genética , Synechocystis/metabolismo
18.
Antimicrob Agents Chemother ; 66(7): e0019822, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35708323

RESUMO

In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 µM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC50). When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to remdesivir resistance.


Assuntos
COVID-19 , SARS-CoV-2 , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Alanina/análogos & derivados , Alanina/metabolismo , Antivirais/química , COVID-19/tratamento farmacológico , Humanos
19.
Biol Trace Elem Res ; 200(11): 4726-4732, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35478087

RESUMO

Lead acetate-motivated oxidative stress can affect all organ systems, particularly the liver. Glutamine (Gln) has both antioxidant and chelating properties. Therefore, we investigated for the first time the effect of Gln on the biochemical and histopathological alternations in a rat model of lead toxicity. Thirty-two rats were divided into four groups (eight rats in each): untreated normal, lead poisoning, and two similar groups receiving Gln (0.1% in drinking water for 4 weeks). To induce lead poisoning, rats received 50 mg/L lead acetate in drinking water for 4 weeks. Oxidative stress indices (total glutathione, the ratio of reduced glutathione to oxidized glutathione, advanced protein oxidation products, malondialdehyde, and ferric ion reducing power) and inflammatory markers (hepatic nuclear factor-kß expression, interleukin 1ß level, and myeloperoxidase activity) were measured. Furthermore, biochemical markers of hepatotoxicity (alanine transaminase, aspartate transaminase, alkaline phosphatase, gamma-glutamyl transpeptidase, total bilirubin, total protein, albumin, and globulins) were measured. Histopathological examination evaluated lead-induced liver damage. The treatment compensated lead-induced biochemical and histopathological alternations in rat liver. Furthermore, it decreased lead acetate level, the NF-kß gene expression, oxidative stress, and inflammatory markers. Moreover, the treatment elevated total glutathione and reduced glutathione in the sera and liver homogenates of treated groups (p < 0.001). Glutamine could protect the liver against lead intoxication via antioxidant, anti-inflammatory, and chelating properties. In addition, its downregulating effect on the hepatic NF-kß signaling pathway confirms its hepatoprotective activity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Água Potável , Alanina/metabolismo , Alanina/farmacologia , Albuminas/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Aspartato Aminotransferases , Bilirrubina , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Glutamina/metabolismo , Glutamina/farmacologia , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Interleucina-1beta/metabolismo , Chumbo/metabolismo , Fígado/metabolismo , Malondialdeído/metabolismo , Estresse Oxidativo , Peroxidase/metabolismo , Ratos , gama-Glutamiltransferase
20.
Protein Sci ; 31(5): e4303, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35481644

RESUMO

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT-Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT-Mi. An in-depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT-Ma and AGT-Mi. Finally, co-immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein-protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well-defined fitness window, thus expanding the adaptability potential of the protein.


Assuntos
Alanina , Transaminases , Alanina/metabolismo , Alelos , Mutação , Transaminases/química
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