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Journal of Structural Biology Jan 2020Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis...
Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.
Topics: Allosteric Regulation; Asparagine; Campylobacter jejuni; Hydro-Lyases; Protein Structure, Quaternary
PubMed: 31678256
DOI: 10.1016/j.jsb.2019.107409 -
Journal of Computational Chemistry Aug 2019The mechanism used by the ubiquitin-conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born-Oppenheimer molecular...
The mechanism used by the ubiquitin-conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born-Oppenheimer molecular dynamics, single point quantum mechanics/molecular mechanics energies, and classical molecular dynamics. These simulations support a long-held hypothesis and show that Ubc13-catalyzed ubiquitination uses a stepwise, nucleophilic attack mechanism. Furthermore, they show that the first step-the formation of a tetrahedral, zwitterionic intermediate-is rate limiting. However, these simulations contradict another popular hypothesis that supposes that the negative charge on the intermediate is stabilized by a highly conserved asparagine (Asn79 in Ubc13). Instead, calculated reaction profiles of the N79A mutant illustrate how charge stabilization actually increases the barrier to product formation. Finally, an alternate role for Asn79 is suggested by simulations of wild-type, N79A, N79D, and H77A Ubc13: it stabilizes the motion of the electrophile prior to the reaction, positioning it for nucleophilic attack. © 2019 Wiley Periodicals, Inc.
Topics: Asparagine; Biocatalysis; Molecular Dynamics Simulation; Molecular Structure; Quantum Theory; Substrate Specificity; Ubiquitin-Conjugating Enzymes; Ubiquitination
PubMed: 31070815
DOI: 10.1002/jcc.25852 -
Chemical Communications (Cambridge,... Apr 2006A simple and efficient synthesis of N-linked glycoamino acids and glycopeptides from deprotected sugars using the Staudinger reaction.
A simple and efficient synthesis of N-linked glycoamino acids and glycopeptides from deprotected sugars using the Staudinger reaction.
Topics: Amino Acid Sequence; Asparagine; Glycosylation; Molecular Structure
PubMed: 16550280
DOI: 10.1039/b515472c -
Cell Chemical Biology Apr 2019The glycan ligands recognized by Siglecs, influenza viruses, and galectins, as well as many plant lectins, are not well defined. To explore their binding to asparagine...
The glycan ligands recognized by Siglecs, influenza viruses, and galectins, as well as many plant lectins, are not well defined. To explore their binding to asparagine (Asn)-linked N-glycans, we synthesized a library of isomeric multiantennary N-glycans that vary in terminal non-reducing sialic acid, galactose, and N-acetylglucosamine residues, as well as core fucose. We identified specific recognition of N-glycans by several plant lectins, human galectins, influenza viruses, and Siglecs, and explored the influence of sialic acid linkages and branching of the N-glycans. These results show the unique recognition of complex-type N-glycans by a wide variety of glycan-binding proteins and their abilities to distinguish isomeric structures, which provides new insights into the biological roles of these proteins and the uses of lectins in biological applications to identify glycans.
Topics: Animals; Asparagine; Binding Sites; Galectins; Humans; Isomerism; Orthomyxoviridae; Plant Lectins; Plants; Polysaccharides; Protein Binding; Proteins; Sialic Acid Binding Immunoglobulin-like Lectins
PubMed: 30745240
DOI: 10.1016/j.chembiol.2019.01.002 -
The Journal of Biological Chemistry Apr 2019The P-type ATPase protein family includes, in addition to ion pumps such as Ca-ATPase and Na,K-ATPase, also phospholipid flippases that transfer phospholipids between...
The P-type ATPase protein family includes, in addition to ion pumps such as Ca-ATPase and Na,K-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 ( V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein.
Topics: Adenosine Triphosphatases; Amino Acid Substitution; Animals; Asparagine; Cattle; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Mutation, Missense; Phospholipid Transfer Proteins; Phosphorylation
PubMed: 30760526
DOI: 10.1074/jbc.RA118.007240 -
Nature Jun 1953
Topics: Asparagine; Blood; Humans; Plasma
PubMed: 13072518
DOI: 10.1038/1711123a0 -
Plant Signaling & Behavior Dec 2023Soybean, a vital protein-rich crop, offers bioactivity that can mitigate various chronic human diseases. Nonetheless, soybean breeding poses a challenge due to the...
Soybean, a vital protein-rich crop, offers bioactivity that can mitigate various chronic human diseases. Nonetheless, soybean breeding poses a challenge due to the negative correlation between enhanced protein levels and overall productivity. Our previous studies demonstrated that applying gaseous phytohormone, ethylene, to soybean leaves significantly boosts the accumulation of free amino acids, particularly asparagine (Asn). Current studies also revealed that ethylene application to soybeans significantly enhanced both essential and non-essential amino acid contents in leaves and stems. Asn plays a crucial role in ammonia detoxification and reducing fatigue. However, the molecular evidence supporting this phenomenon remains elusive. This study explores the molecular mechanisms behind enhanced Asn accumulation in ethylene-treated soybean leaves. Transcriptional analysis revealed that ethylene treatments to soybean leaves enhance the transcriptional levels of key genes involved in Asn biosynthesis, such as aspartate aminotransferase (AspAT) and Asn synthetase (ASN), which aligns with our previous observations of elevated Asn levels. These findings shed light on the role of ethylene in upregulating Asn biosynthetic genes, subsequently enhancing Asn concentrations. This molecular insight into amino acid metabolism regulation provides valuable knowledge for the metabolic farming of crops, especially in elevating nutraceutical ingredients with non-genetic modification (GM) approach for improved protein content.
Topics: Amino Acids; Asparagine; Ethylenes; Seeds; Glycine max
PubMed: 38019725
DOI: 10.1080/15592324.2023.2287883 -
Asparagine-Glucose Amadori Compounds: Formation, Characterization, and Analysis in Dry Jujube Fruit.Journal of Agricultural and Food... Apr 2024Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid...
Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid chromatography-mass spectroscopy. With the study on the effect of the reaction temperature, pH values, and reaction time, the ideal reaction condition for accumulation of Asn-Glc-ARP was determined at 100 °C for 40 min under pH 7. Asparagine (Asn) was prone to degrade from Asn-Glc-ARP in alkaline pH values within a lower temperature range, while in an acidic environment with high temperatures, deamidation of Asn-Glc-ARP to Asp-Glc-ARP (Amadori rearrangement products of aspartic acid with glucose) was displayed as the dominant pathway. The deamidation reaction on the side chain of the amide group took place at Asn-Glc-ARP and transferred it into the hydroxyl group, forming Asp-Glc-ARP at the end. Considering that lyophilization as pretreatment led to limited water activity, a single aspartic acid was not deamidated from Asn directly nor did it degrade from Asp-Glc-ARP even at 120 °C. The degradation of Asn-Glc-ARP through tandem mass spectrometry (MS/MS) analysis showed the obvious fragment ion at / 211, indicating that the stable oxonium ion formed during fragmentation. The structure of Asn-Glc-ARP was proposed as 1-deoxy-1-l-asparagino-d-fructose after separation and purification. Also, the content of Asn-Glc-ARP within dry jujube fruit (HeTianYuZao) was quantitated as high as 8.1 ± 0.5 mg/g.
Topics: Asparagine; Glucose; Tandem Mass Spectrometry; Maillard Reaction; Aspartic Acid; Plant Extracts; Ziziphus
PubMed: 38502793
DOI: 10.1021/acs.jafc.4c00526 -
International Review of Cell and... 2022Metabolic rewiring is a characteristic hallmark of cancer cells. This phenomenon sustains uncontrolled proliferation and resistance to apoptosis by increasing nutrients... (Review)
Review
Metabolic rewiring is a characteristic hallmark of cancer cells. This phenomenon sustains uncontrolled proliferation and resistance to apoptosis by increasing nutrients and energy supply. However, reprogramming comes together with vulnerabilities that can be used against tumor and can be applied in targeted therapy. In the last years, the genetic background of tumors has been identified thoroughly and new therapies targeting those mutations tested. Nevertheless, we propose that targeting the phenotype of cancer cells could be another way of treatment aiming to avoid drug resistance and non-responsiveness of cancer patients. Amino acid metabolism is part of the altered processes in cancer cells. Amino acids are building blocks and also sensors of signaling pathways regulating main biological processes. In this comprehensive review, we described four amino acids (asparagine, arginine, methionine, and cysteine) which have been actively investigated as potential targets for anti-tumor therapy. Asparagine depletion is successfully used for decades in the treatment of acute lymphoblastic leukemia and there is a strong implication to apply it to other types of tumors. Arginine auxotrophic tumors are great candidates for arginine-starvation therapy. Higher requirement for essential amino acids such as methionine and cysteine point out promising targetable weaknesses of cancer cells.
Topics: Humans; Asparagine; Cysteine; Neoplasms; Amino Acids; Arginine; Methionine
PubMed: 36283767
DOI: 10.1016/bs.ircmb.2022.08.001 -
Biochemistry Dec 2017Coiled coils (CCs) are among the best-understood protein folds. Nonetheless, there are gaps in our knowledge of CCs. Notably, CCs are likely to be structurally more...
Coiled coils (CCs) are among the best-understood protein folds. Nonetheless, there are gaps in our knowledge of CCs. Notably, CCs are likely to be structurally more dynamic than often considered. Here, we explore this in an abundant class of CCs, parallel dimers, focusing on polar asparagine (Asn) residues in the hydrophobic interface. It is well documented that such inclusions discriminate between different CC oligomers, which has been rationalized in terms of whether the Asn can make side-chain hydrogen bonds. Analysis of parallel CC dimers in the Protein Data Bank reveals a variety of Asn side-chain conformations, but not all of these make the expected inter-side-chain hydrogen bond. We probe the structure and dynamics of a de novo-designed coiled-coil homodimer, CC-Di, by multidimensional nuclear magnetic resonance spectroscopy, including model-free dynamical analysis and relaxation-dispersion experiments. We find dynamic exchange on the millisecond time scale between Asn conformers with the side chains pointing into and out of the core. We perform molecular dynamics simulations that are consistent with this, revealing that the side chains are highly dynamic, exchanging between hydrogen-bonded-paired conformations in picoseconds to nanoseconds. Combined, our data present a more dynamic view for Asn at CC interfaces. Although inter-side-chain hydrogen bonding states are the most abundant, Asn is not always buried or engaged in such interactions. Because interfacial Asn residues are key design features for modulating CC stability and recognition, these further insights into how they are accommodated within CC structures will aid their predictive modeling, engineering, and design.
Topics: Amino Acid Sequence; Asparagine; Computer Simulation; Databases, Protein; Dimerization; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Protein Conformation; Protein Structure, Secondary; Proteins
PubMed: 29166010
DOI: 10.1021/acs.biochem.7b00848