Identification of the peptide epimerase MslH responsible for d-amino acid introduction at the C-terminus of ribosomal peptides.
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ABSTRACT:
SUBMITTER: Feng Z
PROVIDER: S-EPMC8179263 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Biologically uncommon d-aspartate (d-Asp) residues have been shown to accumulate in proteins associated with age-related human disorders, such as cataract and Alzheimer disease. Such d-Asp-containing proteins are unlikely to be broken down completely because metabolic enzymes recognize only proteins or peptides composed exclusively of l-amino acids. Therefore, undigested d-Asp-containing peptides may exist in blood and, if detectable, may be a useful biomarker for associated diseases. In this study, we investigated d-amino acid-containing peptides in adult human serum by a qualitative d-amino acid analysis based on a diastereomer method and LC-MS/MS method. As a result, two d-Asp-containing peptides were detected in serum, both derived from the fibrinogen ?-chain, a glycoprotein that helps in the formation of blood clots. One of the peptides was fibrinopeptide B, which prevents fibrinogen from forming polymers of fibrin, and the other was same peptide with C-terminal Arginine missing. To our knowledge, this is the first report of the presence of d-amino acid-containing peptides in serum and the approach described will provide a new direction on the serum proteome and fragmentome.
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Project description:Diketopiperazine (DKP) formation is an important degradation pathway for peptides and proteins. It can occur during synthesis and storage in either solution or the solid state. The kinetics of peptide cleavage through DKP formation have been analyzed for the model peptides Xaa1-Pro2-Gly4-Lys7 [Xaa = Gln, Glu, Lys, Ser, Phe, Trp, Tyr, Cha (β-cyclohexylalanine), Aib (α-aminoisobutyric acid), Gly, and Val] at multiple elevated temperatures in ethanol with ion mobility spectrometry-mass spectrometry (IMS-MS). When Xaa is an amino acid with a charged or polar side chain, degradation is relatively fast. When Xaa is an amino acid with a nonpolar alkyl side chain, the peptide is relatively stable. For these peptides, a bulky group on the α carbon speeds up dissociation, but the kinetic effects vary in a complicated manner for bulky groups on the β or γ carbon. Peptides where Xaa has a nonpolar aromatic side chain show moderate dissociation rates. The stability of these peptides is a result of multiple factors. The reaction rate is enhanced by (1) the stabilization of the late transition state through the interaction of an aromatic ring with the nascent DKP ring or lowering the activation energy of nucleophilic attack intermediate state through polar or charged residues and (2) the preference of the cis proline bond favored by the aromatic N-terminus. The number of unseen intermediates and transition state thermodynamic values are derived for each peptide by modeling the kinetics data. Most of the transition states are entropically favored (ΔS⧧ ∼ -5 to +31 J·mol-1·K-1), and all are enthalpically disfavored (ΔH⧧ ∼ 93 to 109 kJ·mol-1). The Gibbs free energy of activation is similar for all of the peptides studied here (ΔG⧧ ∼ 90-99 kJ·mol-1).
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Project description:BackgroundThe application of in vitro translation to the synthesis of unnatural peptides may allow the production of extremely large libraries of highly modified peptides, which are a potential source of lead compounds in the search for new pharmaceutical agents. The specificity of the translation apparatus, however, limits the diversity of unnatural amino acids that can be incorporated into peptides by ribosomal translation. We have previously shown that over 90 unnatural amino acids can be enzymatically loaded onto tRNA.Methodology/principal findingsWe have now used a competition assay to assess the efficiency of tRNA-aminoacylation of these analogs. We have also used a series of peptide translation assays to measure the efficiency with which these analogs are incorporated into peptides. The translation apparatus tolerates most side chain derivatives, a few alpha,alpha disubstituted, N-methyl and alpha-hydroxy derivatives, but no beta-amino acids. We show that over 50 unnatural amino acids can be incorporated into peptides by ribosomal translation. Using a set of analogs that are efficiently charged and translated we were able to prepare individual peptides containing up to 13 different unnatural amino acids.Conclusions/significanceOur results demonstrate that a diverse array of unnatural building blocks can be translationally incorporated into peptides. These building blocks provide new opportunities for in vitro selections with highly modified drug-like peptides.
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Project description:Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative ?-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative ?-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no ?-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 ?mol·min(-1)·mg(-1), respectively, and those for d-allo-isoleucine were 13.2 mM and 286 ?mol·min(-1)·mg(-1), respectively. Hydroxylamine and other inhibitors of pyridoxal 5'-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5'-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.
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Project description:Halogenation plays a significant role in the activity of the glycopeptide antibiotics (GPAs), although up until now the timing and therefore exact substrate involved was unclear. Here, we present results combined from in vivo and in vitro studies that reveal the substrates for the halogenase enzymes from GPA biosynthesis as amino acid residues bound to peptidyl carrier protein (PCP)-domains from the non-ribosomal peptide synthetase machinery: no activity was detected upon either free amino acids or PCP-bound peptides. Furthermore, we show that the selectivity of GPA halogenase enzymes depends upon both the structure of the bound amino acid and the PCP domain, rather than being driven solely via the PCP domain. These studies provide the first detailed understanding of how halogenation is performed during GPA biosynthesis and highlight the importance and versatility of trans-acting enzymes that operate during peptide assembly by non-ribosomal peptide synthetases.
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Project description:Histone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vitro. However, the H3.Y residues responsible for its stable nucleosome incorporation have not been identified yet. In the present study, we performed comprehensive mutational analyses of H3.Y, and determined that the H3.Y C-terminal region including amino acid residues 124-135 is responsible for its stable association with DNA. Among the H3.Y C-terminal residues, the H3.Y Met124 residue significantly contributed to the stable DNA association with the H3.Y-H4 tetramer. The H3.Y M124I mutation substantially reduced the H3.Y-H4 association in the nucleosome. In contrast, the H3.Y K42R mutation affected the nucleosome stability less, although it contributes to the flexible DNA ends of the nucleosome. Therefore, these H3.Y-specific residues, Lys42 and Met124, play different and specific roles in nucleosomal DNA relaxation and stable nucleosome formation, respectively, in chromatin.
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Project description:Eukaryotic ribosomal protein L13a is a member of the conserved universal ribosomal uL13 protein family. Structurally, L13a is distinguished from its prokaryotic counterparts by the presence of an ?55 amino acid-long carboxy-terminal ?-helical extension. The importance of these evolved residues in the carboxy-terminal extension for mammalian ribosome biogenesis as well as L13a's extraribosomal function in GAIT (? interferon-activated inhibitor of translation) complex-mediated translation silencing during inflammation is not understood. Here, we present biochemical analyses of L13a mutant variants identifying several mutually exclusive amino acid residues in the eukaryote-specific carboxy-terminal extension of human L13a (Tyr149-Val203) important for ribosomal incorporation and translational silencing. Specifically, we show that mutation of Arg169, Lys170, and Lys171 to Ala abrogate GAIT-mediated translational silencing, but not L13a incorporation into ribosomes. Moreover, we show that the carboxy-terminal helix alone can silence translation of GAIT element-containing mRNAs in vitro. We also show through cellular immunofluorescence experiments that nuclear but not nucleolar localization of L13a is resistant to extensive amino acid alterations, suggesting that multiple complex nuclear import signals are present within this protein. These studies provide new insights into L13a structure and its ribosomal and extraribosomal functions in model human cells.
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Project description:There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues. Here, we demonstrate that the wild-type aerolysin nanopore enables the size-discrimination of several short uniformly charged homopeptides, mixed in solution, with a single amino acid resolution. Our system is very sensitive, allowing detecting and characterizing a few dozens of peptide impurities in a high purity commercial peptide sample, while conventional analysis techniques fail to do so.
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Project description:The lasso peptide MS-271 is a ribosomally synthesized and post-translationally modified peptide (RiPP) consisting of 21 amino acids with D-tryptophan at the C-terminus, and is derived from the precursor peptide MslA. MslH, encoded in the MS-271 biosynthetic gene cluster (msl), catalyzes the epimerization at the Cα center of the MslA C-terminal Trp21, leading to epi-MslA. The detailed catalytic process, including the catalytic site and cofactors, has remained enigmatic. Herein, based on X-ray crystallographic studies in association with MslA core peptide analogues, we show that MslH is a metallo-dependent peptide epimerase with a calcineurin-like fold. The crystal structure analysis, followed by site-directed mutagenesis, docking simulation, and ICP-MS studies demonstrate that MslH employs acid/base chemistry to facilitate the reversible epimerization of the C-terminal Trp21 of MslA, by utilizing two pairs of His/Asp catalytic residues that are electrostatically tethered to a six-coordination motif with a Ca(II) ion via water molecules.
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Project description:A new method for enhancing peptide ion identification in proteomics analyses using ion mobility data is presented. Ideally, direct comparisons of experimental drift times (t(D)) with a standard mobility database could be used to rank candidate peptide sequence assignments. Such a database would represent only a fraction of sequences in protein databases and significant difficulties associated with the verification of data for constituent peptide ions would exist. A method that employs intrinsic amino acid size parameters to obtain ion mobility predictions that can be used to rank candidate peptide ion assignments is proposed. Intrinsic amino acid size parameters have been determined for doubly charged peptide ions from an annotated yeast proteome. Predictions of ion mobilities using the intrinsic size parameters are more accurate than those obtained from a polynomial fit to t(D) versus molecular weight data. More than a 2-fold improvement in prediction accuracy has been observed for a group of arginine-terminated peptide ions 12 residues in length. The use of this predictive enhancement as a means to aid peptide ion identification is discussed, and a simple peptide ion scoring scheme is presented.
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