dehydrobutyrine and dehydroalanine

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)-Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha-(E)-Dhb. It displays narrow-spectrum activity against vancomycin-resistant Enterococcus faecium. In-vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria.

Topics: Alanine; Aminobutyrates; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Proteins; Drug Evaluation, Preclinical; Drug Resistance, Bacterial; Enterococcus faecium; Multigene Family; Nuclear Magnetic Resonance, Biomolecular; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Stereoisomerism; Streptomyces

Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such "elimination damage" generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically.

Topics: Alanine; Aminobutyrates; Animals; Antimicrobial Cationic Peptides; Female; Glutathione; HEK293 Cells; Humans; Male; MAP Kinase Kinase 1; Membrane Proteins; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase Kinases; Phosphate-Binding Proteins; Phosphorylation; Protein Domains; Proteome; Receptors, G-Protein-Coupled; Sulfides

Bacterial phosphothreonine lyases, or phospholyases, catalyze a unique post-translational modification that introduces dehydrobutyrine (Dhb) or dehydroalanine (Dha) in place of phosphothreonine or phosphoserine residues, respectively. We report the use of a phospha-Michael reaction to label proteins and peptides modified with Dha or Dhb. We demonstrate that a nucleophilic phosphine probe is able to modify Dhb-containing proteins and peptides that were recalcitrant to reaction with thiol or amine nucleophiles under mild aqueous conditions. Furthermore, we used this reaction to detect multiple Dhb-modified proteins in mammalian cell lysates, including histone H3, a previously unknown target of phospholyases. This method should prove useful for identifying new phospholyase targets, profiling the biomarkers of bacterial infection, and developing enzyme-mediated strategies for bioorthogonal labeling in living cells.

Topics: Alanine; Amines; Aminobutyrates; Bacteria; Bacterial Infections; Biomarkers; Histones; Humans; Lyases; Phosphines; Phosphothreonine; Protein Processing, Post-Translational; Sulfhydryl Compounds

Expansion of the genetic code with unnatural amino acids (Uaas) has significantly increased the chemical space available to proteins for exploitation. Due to the inherent limitation of translational machinery and the required compatibility with biological settings, function groups introduced via Uaas to date are restricted to chemically inert, bioorthogonal, or latent bioreactive groups. To break this barrier, here we report a new strategy enabling the specific incorporation of biochemically reactive amino acids into proteins. A latent bioreactive amino acid is genetically encoded at a position proximal to the target natural amino acid; they react via proximity-enabled reactivity, selectively converting the latter into a reactive residue in situ. Using this Genetically Encoded Chemical COnversion (GECCO) strategy and harnessing the sulfur-fluoride exchange (SuFEx) reaction between fluorosulfate-l-tyrosine and serine or threonine, we site-specifically generated the reactive dehydroalanine and dehydrobutyrine into proteins. GECCO works both inter- and intramolecularly, and is compatible with various proteins. We further labeled the resultant dehydroalanine-containing protein with thiol-saccharide to generate glycoprotein mimetics. GECCO represents a new solution for selectively introducing biochemically reactive amino acids into proteins and is expected to open new avenues for exploiting chemistry in live systems for biological research and engineering.

Topics: Alanine; Aminobutyrates; Models, Molecular; Protein Engineering; Protein Structure, Secondary

Dehydroalanine (DHA) and dehydrobutyrine (DHB) intermediates, formed through β-elimination, induce protein irreversible glutathionylation and protein-protein crosslinking in human lens fiber cells. In total, irreversible glutathionylation was detected on 52 sites including cysteine, serine and threonine residues in 18 proteins in human lenses. In this study, the levels of GSH modification on three serine residues and four cysteine residues located in seven different lens proteins isolated from different regions and different aged lenses were quantified. The relative levels of modification (modified/nonmodified) were site-specific and age-related, ranging from less than 0.05% to about 500%. The levels of modification on all of the sites quantified in the lens cortex increased with age and GSH modification also increased from cortex to outer nucleus region suggesting an age-related increase of modification. The levels of modification on sites located in stable regions of the proteins such as Cys117 of βA3, Cys80 of βB1 and Cys27 of γS, continued increasing in inner nucleus, but modification on sites located in regions undergoing degradation with age decreased in the inner nucleus suggesting GSH modified proteins were more susceptible to further modification. Irreversible GSH modification in cataract lenses was typically higher than in age-matched normal lenses, but the difference did not reach statistical significance for a majority of sites, with the exception Cys117 of βA3 crystallin in WSF. Except for S59 of αA and αB crystallins, GSH modification did not induce protein insolubility suggesting a possible role for this modification in protection from protein-protein crosslinking.

Topics: Adolescent; Alanine; Aminobutyrates; Cataract; Cellular Senescence; Chromatography, Liquid; Crystallins; Cysteine; Glutathione; Humans; Lens, Crystalline; Middle Aged; Protein Processing, Post-Translational; Serine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfides; Threonine; Tissue Donors; Young Adult

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) are remarkably versatile non-canonical amino acids often found in antimicrobial peptides. This work presents the selective modification of Dha and Dhb in antimicrobial peptides through photocatalytic activation of organoborates under the influence of visible light. Ir(dF(CF

Topics: Alanine; Aminobutyrates; Borates; Catalysis; Coordination Complexes; Iridium; Light; Nisin; Oxidation-Reduction; Photochemical Processes; Ruthenium; Thiostrepton

Old long-lived proteins contain dehydroalanine (Dha) and dehydrobutyrine (Dhb), two amino acids engendered by dehydration of serines and threonines, respectively. Although these residues have a suspected role in protein cross-linking and aggregation, their direct implication has yet to be determined. Here, we have taken advantage of the ability of the enteropathogen

Topics: Alanine; Aminobutyrates; Animals; Antibodies; Bacterial Proteins; Caco-2 Cells; Carbon-Oxygen Lyases; Cell Line; Extracellular Signal-Regulated MAP Kinases; HeLa Cells; Humans; MAP Kinase Signaling System; Mice; p38 Mitogen-Activated Protein Kinases; Protein Binding; Proteomics; Shigella; Substrate Specificity; Threonine; Type III Secretion Systems

Herein the synthesis of two nisin AB dicarba analogs is described, focusing on amino acid modifications at positions 2 and 5. The nisin mimics were synthesized by a combination of solid phase synthesis of the linear peptides, followed by macrocyclization via ring-closing metathesis and fragment assembly by means of solution phase chemistry. The two N-terminal nisin AB-fragment mimics contain either the native dehydrobutyrine (Dhb)/dehydroalanine (Dha) amino acid residues or alanine at position 2 and 5, respectively. The native dehydrobutyrine at position 2 and dehydroalanine at position 5 were introduced as their precursors, namely threonine and serine, respectively, and subsequent dehydration was carried out by EDCI/CuCl as the condensing agent. Both AB-fragment mimics were analyzed in a lipid II binding assay and it was found that the Ala2/Ala5 AB-mimic (2) showed a reduced activity, while the Dhb2/Dha5 AB-mimic (3) was as active as the native AB-fragment (1).

Topics: Alanine; Amino Acid Sequence; Aminobutyrates; Anti-Bacterial Agents; Molecular Docking Simulation; Molecular Sequence Data; Nisin; Unilamellar Liposomes; Uridine Diphosphate N-Acetylmuramic Acid

Nonenzymatic post-translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC-tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty-four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β-elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein-protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2- & βB2-, βA4- & βA3-, γS- & βB1-, and βA4- & βA4-crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long-lived proteins and that DHA- and DHB-mediated protein crosslinking may be the source of the long-sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long-lived proteins of other tissues leading to protein aggregation diseases.

Topics: Adult; Aged, 80 and over; Alanine; Amino Acid Sequence; Aminobutyrates; Cell Nucleus; Cross-Linking Reagents; Crystallins; Glutathione; Humans; Lens, Crystalline; Middle Aged; Molecular Sequence Data; Peptides; Protein Processing, Post-Translational; Serine; Tandem Mass Spectrometry; Threonine; Time Factors

Oxazole ring occurs in numerous natural peptides, but conformational properties of the amino acid residue containing the oxazole ring in place of the C-terminal amide bond are poorly recognized. A series of model compounds constituted by the oxazole-amino acids occurring in nature, that is, oxazole-alanine (L-Ala-Ozl), oxazole-dehydroalanine (ΔAla-Ozl), and oxazole-dehydrobutyrine ((Z)-ΔAbu-Ozl), was investigated using theoretical calculations supported by FTIR and NMR spectra and single-crystal X-ray diffraction. It was found that the main feature of the studied oxazole-amino acids is the stable conformation β2 with the torsion angles φ and ψ of -150°, -10° for L-Ala-Ozl, -180°, 0° for ΔAla-Ozl, and -120°, 0° for (Z)-ΔAbu-Ozl, respectively. The conformation β2 is stabilized by the intramolecular N-H···N hydrogen bond and predominates in the low polar environment. In the case of the oxazole-dehydroamino acids, the π-electron conjugation that is spread on the oxazole ring and C(α)═C(β) double bond is an additional stabilizing interaction. The tendency to adopt the conformation β2 clearly decreases with increasing the polarity of environment, but still the oxazole-dehydroamino acids are considered to be more rigid and resistant to conformational changes.

Topics: Alanine; Amino Acids; Aminobutyrates; Hydrogen; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Theoretical; Molecular Conformation; Nitrogen; Oxazoles; Solvents; Spectroscopy, Fourier Transform Infrared; Surface Properties; Thermodynamics

Glutathionylation is generally a reversible posttranslational modification that occurs to cysteine residues that have been exposed to reactive oxygen species (P-SSG). This cyclical process can regulate various clusters of proteins, including those involved in critical cellular signaling functions. However, certain conditions can favor the formation of dehydroamino acids, such as 2,3-didehydroalanine (2,3-dehydroalanine, DHA) and 2,3-didehydrobutyrine (2,3-dehydrobutyrine), which can act as Michael acceptors. In turn, these can form Michael adducts with glutathione (GSH), resulting in the formation of a stable thioether conjugate, an irreversible process referred to as nonreducible glutathionylation. This is predicted to be prevalent in nature, particularly in more slowly turning over proteins. Such nonreducible glutathionylation can be distinguished from the more facile cycling signaling processes and is predicted to be of gerontological, toxicological, pharmacological, and oncological relevance. Here, we compare reversible and irreversible glutathionylation.

Topics: Alanine; Aminobutyrates; Enzymes; Gene Expression Regulation, Neoplastic; Glutaredoxins; Glutathione; Glutathione Disulfide; Glutathione Transferase; Humans; Lens, Crystalline; Neoplasms; Peptides; Proteins; Reactive Oxygen Species; Signal Transduction

We have devised a protocol for enzyme-free insertion of dehydroalanine, dehydrobutyrine and thioether crosslinks into translated peptides. In vitro translation using 4-selenalysine and 4-selenoisoleucine as substitutes for lysine and isoleucine yields peptides that can be converted to polycyclic structures using mild chemistry in water. This methodology presents a gateway for exploring the potential of artificial lantipeptides as scaffolds for drug development.

Topics: Alanine; Amino Acid Sequence; Aminobutyrates; Cross-Linking Reagents; Lysine; Molecular Sequence Data; Organoselenium Compounds; Peptide Biosynthesis; Peptides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfides

The lantibiotics are ribosomally synthesized and posttranslationally modified peptide antibiotics containing the thioether crosslinks lanthionine (Lan) and 3-methyllanthionine (MeLan) and typically also the dehydroamino acids dehydroalanine (Dha) and (Z)-dehydrobutyrine (Dhb). These modifications are formed by dehydration of Ser/Thr residues to produce the Dha and Dhb structures, and subsequent conjugate additions of Cys residues onto the unsaturated amino acids to form thioether rings (Lan and MeLan). Several of the enzymatic reactions involved in lantibiotic biosynthesis have been reconstituted in vitro in recent years and these systems as well as a general overview of lantibiotic biosynthesis are discussed in this chapter.

Topics: Alanine; Aminobutyrates; Bacteriocins; Molecular Structure; Sulfides

Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for enzymatic processing. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481.

Topics: 5' Untranslated Regions; Alanine; Amino Acid Sequence; Aminobutyrates; Bacteriocins; Conserved Sequence; DNA Primers; Enzymes; Gene Expression; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Sequence Alignment; Sulfides

Nisin variants and fragments were reacted with glutathione, and the products of the reactions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography/mass spectrometry (LC-MS). Reactions between glutathione and either [Ala5]nisin or [Ala33]nisin resulted in products with two glutathione molecules conjugated to one nisin variant molecule. Only one glutathione molecule was added to [Ala5,Ala33]nisin. Fragmentation of the nisin molecule resulted in nisin 1-12, nisin 1-20, and nisin 1-32 fragments. Each fragment retained two dehydro residues, which subsequently underwent reaction with glutathione. The data indicated that the dehydroalanine residues of nisin are sites of addition for glutathione. Such addition renders the nisin molecule inactive.

Topics: Alanine; Amino Acid Sequence; Aminobutyrates; Anti-Bacterial Agents; Chromatography, High Pressure Liquid; Food Preservatives; Glutathione; Mass Spectrometry; Molecular Sequence Data; Nisin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Topics: Alanine; Amino Acid Sequence; Aminobutyrates; Molecular Sequence Data; Peptides