Incorporation Of Tyrosine Research Articles

Merging Flow Synthesis and Enzymatic Maturation to Expand the Chemical Space of Lasso Peptides.

Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by in vitro transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogues of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analogue containing three d-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and the improvement of therapeutic properties.

Site-specific encoding of photoactivity and photoreactivity into antibody fragments

Design of biomolecules that perform two or more distinct functions in response to light remains challenging. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12–EGFR binding affinity in the absence of light. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR.

Incorporation of tyrosine in the Co/MCM-41: Green and efficient peroxidase mimetic

Tyrosine (Tyr) was incorporated in the Co/MCM-41 channels by adding template-containing Co/MCM-41 and Tyr solution via a simple green approach. The prepared material was characterized using UV–vis, FT-IR, XRD, TGA-DTG, EDX, and N2 adsorption/desorption analytical techniques. For comparison, Co-Tyr/[CTA]MCM-41 (CTA: Cetyltrimethylammonium bromide) was also synthesized by mixing a solution of Co-Tyr with [CTA]MCM-41. The prepared samples were used as a peroxidase mimetic in the presence of H2O2. Co-Tyr/[CTA]MCM-41 prepared by the first method was found to be highly active for the oxidation of 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB), following the typical Michaelis-Menten kinetics and exhibiting the excellent peroxidase-like catalytic performance. This high activity was due to the phenol hydroxyl group of the Tyr, the presence of CTA+ cations, and hydrophobicity induced by the organic template.

Engineering SHP2 Phosphatase for Optical Control.

Signal transduction pathways are responsible for maintaining cellular functions, including proliferation, differentiation, apoptosis, and cell cycle progression. These pathways are maintained through the propagation of phosphorylation signals by protein kinases, as well as the removal of phosphorylation signals by protein phosphatases. Depending on the context, post-translational modification could have either a positive or negative effect on a signaling pathway. Intricate networks of positive and negative regulators offer a challenging target for the dissection of cell signaling mechanisms, particularly regarding the more subtle dampening of signal transduction through phosphatases. We report the development of two complimentary methods for the optical control of a complex phosphatase: SH2 domain-containing protein tyrosine phosphatase-2 (SHP2). We investigated controlling the catalytic function of SHP2 through (1) site-specific incorporation of a caged tyrosine for light activation of catalytic activity for the control of an essential substrate binding residue and (2) site-specific incorporation of a caged lysine at a conserved residue within an allosteric pocket for the control of SHP2 binding partner docking sites. These methods are generalizable to proteins bearing either a protein tyrosine phosphatase (PTP) catalytic domain or an SH2 domain, including SHP1, PTP family phosphatases, and a diverse range of SH2 domain-containing proteins.

Thwarting protein synthesis leads to malaria parasite paralysis

Inhibiting translation presents a tantalizing strategy to tackle the most virulent human malaria parasite. Xie et al. disclose a compound that binds selectively to Plasmodium falciparum tyrosine aminoacyl-tRNA synthetase, preventing the incorporation of tyrosine into nascent proteins and paving the way for a new generation of safe, effective antimalarials.

Light‐Triggered In Situ Biosynthesis of Artificial Melanin for Skin Protection (Adv. Sci. 7/2022)

Artificial Melanosome for Transdermal Delivery In article number 2103503 by Uk-Jae Lee, Nathaniel S. Hwang, Byung-Gee Kim, and co-workers, light-triggered in situ biosynthesis of artificial melanin in native skin is proposed and demonstrated using photoactivatable tyrosinase. Photoactivatable tyrosinase is synthesized with site-specific incorporation of o-nitrobenzyl tyrosine and then encapsulated into lipid nanoparticles as an artificial melanosome for transdermal delivery. This biomimetic system can recapitulate the biosynthetic analogs of naturally occurring melanin.

Directed Evolution Pipeline for the Improvement of Orthogonal Translation Machinery for Genetic Code Expansion at Sense Codons.

The expansion of the genetic code beyond a single type of noncanonical amino acid (ncAA) is hindered by inefficient machinery for reassigning the meaning of sense codons. A major obstacle to using directed evolution to improve the efficiency of sense codon reassignment is that fractional sense codon reassignments lead to heterogeneous mixtures of full-length proteins with either a ncAA or a natural amino acid incorporated in response to the targeted codon. In stop codon suppression systems, missed incorporations lead to truncated proteins; improvements in activity may be inferred from increased protein yields or the production of downstream reporters. In sense codon reassignment, the heterogeneous proteins produced greatly complicate the development of screens for variants of the orthogonal machinery with improved activity. We describe the use of a previously-reported fluorescence-based screen for sense codon reassignment as the first step in a directed evolution workflow to improve the incorporation of a ncAA in response to the Arg AGG sense codon. We first screened a library with diversity introduced into both the orthogonal Methanocaldococcus jannaschii tyrosyl tRNA anticodon loop and the cognate aminoacyl tRNA synthetase (aaRS) anticodon binding domain for variants that improved incorporation of tyrosine in response to the AGG codon. The most efficient variants produced fluorescent proteins at levels indistinguishable from the E. coli translation machinery decoding tyrosine codons. Mutations to the M. jannaschii aaRS that were found to improve tyrosine incorporation were transplanted onto a M. jannaschii aaRS evolved for the incorporation of para-azidophenylalanine. Improved ncAA incorporation was evident using fluorescence- and mass-based reporters. The described workflow is generalizable and should enable the rapid tailoring of orthogonal machinery capable of activating diverse ncAAs to any sense codon target. We evaluated the selection based improvements of the orthogonal pair in a host genomically engineered for reduced target codon competition. Using this particular system for evaluation of arginine AGG codon reassignment, however, E. coli strains with genomes engineered to remove competing tRNAs did not outperform a standard laboratory E. coli strain in sense codon reassignment.

Biosynthesis of the Fusarium Mycotoxin (-)-Sambutoxin.

4-Hydroxy-2-pyridone alkaloids have attracted attention for synthetic and biosynthetic studies due to their broad biological activities and structural diversity. Here, we elucidated the pathway and chemical logic of (-)-sambutoxin (1) biosynthesis. In particular, we uncovered the enzymatic origin of the tetrahydropyran moiety and showed that the p-hydroxyphenyl group is installed via a late-stage, P450-catalyzed oxidation of the phenylalanine-derived side chain rather than via a direct incorporation of tyrosine.

A new path to tyrosine sulfation.

Tyrosine sulfation is a common post-translational modification known to play critical roles in multiple bioprocesses. A cleverly engineered mammalian expanded genetic code now enables the direct co-translational incorporation of tyrosine sulfates into proteins to study their function in cellular contexts.

Effect of citrulline on muscle protein turnover in an in vitro model of muscle catabolism.

Muscle net catabolism, as seen after severe trauma or sepsis or in postoperative situations, is mediated by hormones (e.g., cortisol) and proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-α]). Specific amino acids may be able to limit this muscle mass loss. Citrulline (CIT) stimulates muscle protein synthesis in various situations, but little data exist on hypercatabolic situations and the effects on protein breakdown are unknown. Our aim was to assess the effect of CIT on protein turnover in an in vitro model of muscle hypercatabolism. Myotubes derived from C2C12 myoblasts were treated with 150 nM dexamethasone (DEX), 10 ng/mL TNF-α, or 0.006% ethanol (as control [CON]) for 24 h. Subsequently, myotubes were incubated with or without 5 mM CIT for 6 h. Muscle protein synthesis rate was evaluated by the surface sensing of translation method and by l-[3,5-3H]tyrosine (Tyr) incorporation. The muscle protein breakdown rate was evaluated from Tyr release into culture medium. CIT action was analyzed by non-parametric Kruskal-Wallis and Mann-Whitney tests. CIT treatment significantly increased protein synthesis rates compared with the DEX or TNF-α group (surface sensing of translation method; DEX + CIT versus DEX; P = 0.03 and TNF-α+CIT versus TNF-α; P = 0.05) and significantly decreased protein breakdown rate in the CON and DEX groups (CON + CIT versus CON; P = 0.05 and DEX + CIT versus DEX; P = 0.05). CIT treatment regulated muscle protein turnover in an in vitro model of muscle net catabolism. Exploring the underlying mechanisms would also be of interest.

Tyrosine based cationic acrylates as potent antimicrobial agents against shigellosis.

Incorporation of tyrosine within synthetic macromolecules via the controlled radical polymerization technique is of particular interest due to the presence of a phenolic group in tyrosine imparting high inhibition potential. Tyrosine incorporation is also fascinating from a medicinal point of view since this particular amino acid is present in several natural antimicrobial peptides as a key constituent. This study describes a facile strategy to integrate the naturally occurring proteinogenic amino acid tyrosine within a polyacrylate system via the reversible addition fragmentation chain transfer polymerization technique to construct potent bioactive macromolecules against the microbial strain S. flexneri 2a causing shigellosis. The cationic charge and amphiphilic features inherited from the amino acid tyrosine mimic the function of natural antimicrobial peptides. A library of tyrosine polyacrylates has been synthesized to investigate the effect of molecular weight on antimicrobial activity. The time-dependent morphological switching observed via FESEM measurement revealed membrane rupture followed by leakage of cellular constituents as an effective mechanism of action of the biomimetic tyrosine-based polymers. The details of membrane disintegration and depolarization due to alteration in membrane potential have been evaluated via flow cytometric and fluorescence microscopic measurements. Most importantly, the side chain tyrosine-based polymers show fascinating biocidal features against the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) bacterial strain, the major causative agent of nosocomial infections. The broad spectrum nature of the polymers has been further investigated against the fungus Aspergillus niger and another Gram-negative bacterium, Escherichia coli.

Diversity oriented synthesis of chromene-xanthene hybrids as anti-breast cancer agents

A diverse library of chromene-xanthene hybrids were synthesized through intramolecular Friedel-Crafts reaction of the arenoxy carbinols. Examples include first incorporation of amino acid tyrosine into xanthene skeletons with polar functionalities. A careful structural evaluation revealed that tyrosine crafted chromene-xanthene hybrids exhibited good activities against breast cancer cell lines MCF-7, MDA-MB-231. The lead compound 16 displays significant cell cycle arrest at G1 phase and induces apoptosis in MDA-MB-231 cells.

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence.

Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

Molecular polygamy: The promiscuity of l-phenylalanyl-tRNA-synthetase triggers misincorporation of meta- and ortho-tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells.

In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention.

Characterisation of taxlllaids A-G; natural products from Xenorhabdus indica.

Six new lipodepsipeptides and an additional linear derivative named taxlllaids A-G (1-7) have been identified in the entomopathogenic bacterium Xenorhabdus indica. The structures of the main compounds have been solved by detailed NMR spectroscopic analysis and the structures of minor derivatives were elucidated by a combination of labelling experiments and detailed MS experiments. The absolute configuration of the taxlllaids was deduced by using the advanced Marfey method and analysis of the biosynthesis gene cluster showing the presence of epimerisation domains, which was subsequently proved to be correct by solid-phase peptide synthesis of all taxlllaids. The exchange of a single amino acid in the adenylation domain was shown to be responsible for substrate promiscuity of the third A domain, resulting in the incorporation of leucine, phenylalanine or tyrosine. Bioactivity testing revealed the taxlllaids to be weakly active against Plasmodium falciparum and against a number of eukaryotic cell lines.

The Effect of Alkylphenols on Lobster Shell Hardening

Alkylphenols, anthropogenic estrogenic endocrine disruptors in vertebrates, have been found in lobsters (Homarus americanus) in New England sites. We hypothesize that alkylphenols interfere in the shell hardening during molting. We used an in vitro cuticle bioassay to investigate the effects of 2 alkylphenolic compounds—2,4-bis-(dimethylbenzyl) phenol (compound 3) and bisphenol A (BPA; 4,4#-dihydroxy-2,2-diphenylpropane (also referred to as 4,4#-(propan-2-ylidene) diphenol))—on tyrosine incorporation during the hardening of new cuticle following lobster molting. During sclerotization, both alkylphenols and cold tyrosine competed with C 14 -tyrosine incorporation in a concentration-dependent manner. This process was also phenoloxidase dependent, as treatment with phenylthiourea (PTU; a phenoloxidase inhibitor) significantly decreased C 14 -tyrosine incorporation. We also found that incorporation of C 14 -2,4-bis-(dimethylbenzyl) phenol during the shell hardening process was inhibited by cold alkylphenol, cold tyrosine, or PTU, and competition was concentration dependent. Furthermore, incorporation of tyrosine and derivatives into new cuticle decreased with time after molting from 27% incorporation 1 day after a molt to 6% by 4 days after a molt. In nonmolting cuticles, there was no incorporation of alkylphenol or tyrosine derivatives. When lobsters were injected with 2,4-bis-(dimethylbenzyl) phenol during the premolt stage, it took the shells 12 ± 1 days to harden sufficiently to resist deflection by 5 lb pressure exerted by a pressure gauge, compared with 7 ± 1 days for control shells. Thus, shell hardening is delayed significantly by the presence of 2,4-bis-(dimethylbenzyl) phenol. The effects of this compound on shell hardening may result in lobsters susceptibility to microbial invasion and, therefore, may contribute to the onset of shell disease.

A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine

A novel fluorescent amino acid, L-4-chloromethylcoumarin-6-yl-alanine, was obtained from tyrosine by a Pechmann reaction. The assembly of the heterocyclic ring at the tyrosine side chain could be achieved before or after incorporation of tyrosine into a dipeptide, and amino acid and dipeptide ester conjugates were obtained by coupling to a model N-protected alanine. The behaviour of one of the fluorescent conjugates towards irradiation was studied in a photochemical reactor at different wavelengths (254, 300, 350 and 419 nm). The photoreaction course in methanol/HEPES buffer solution (80:20) was followed by HPLC/UV monitoring. It was found that the novel unnatural amino acid could act as a fluorescent label, due to its fluorescence properties, and, more importantly, as a photoactivable unit, due to the short irradiation times necessary to cleave the ester bond between the model amino acid and the coumarin-6-yl-alanine.

Photorelease of tyrosine from a-carboxy-6-nitroveratryl (aCNV) derivatives

The synthesis of photolabile tyrosine derivatives protected on the phenolic oxygen by the α-carboxy-6-nitroveratryl (αCNV) protecting group is described. The compounds undergo rapid photolysis at wavelengths longer than 300 nm to liberate the corresponding phenol in excellent yield (quantum yield for the deprotection of tyrosine = 0.19). Further protection of caged tyrosine is possible, yielding N-Fmoc protected derivatives suitable for direct incorporation of caged tyrosine in solid-phase peptide synthesis.

Quasi one-dimensional assembly of gold nanoparticles templated by a pH-sensitive peptide amphiphile from silk fibroin

One dimensional (1-D) assembly of gold nanoparticles (AuNPs) plays critical roles in fabricating linear optical and electronic devices. Herein, a novel pH-sensitive template formed by C12-GAGAGAGY, a peptide amphiphile based on the sequence of Bombxy mori silkworm silk fibroin, was employed to generate gold nanoparticles (AuNPs) in situ and direct their aggregation. Due to the incorporation of tyrosine, C12-GAGAGAGY was able to reduce Au3+ to AuNPs and further stabilize them at pH 11 without any external reducing or capping reagents. In addition, these AuNPs could reversibly assemble/disassemble by varying pH due to the pH-sensitive assembly of the template, C12-GAGAGAGY. These templates were cylindrical nanofibers at pH 11 and stacking nanoribbons at pH 4. AuNPs were well-dispersed among the networks formed by cylindrical nanofibers of C12-GAGAGAGY at pH 11, while they aggregated on both sides of the stacking nanoribbons at pH 4. Interestingly, unlike traditional pre-defined templates, the pH-induced assembly of C12-GAGAGAGY and assembly/disassembly of AuNPs occurred simultaneously. These results present us with the potential to utilize such smart peptide amphiphile templates for the fabrication of 1-D inorganic nanostructures with promising applications in nano-scale optical devices.

Probing the in vivo biosynthesis of scytonemin, a cyanobacterial ultraviolet radiation sunscreen, through small scale stable isotope incubation studies and MALDI-TOF mass spectrometry

Scytonemin is a dimeric indole phenolic pigment found in the sheaths of many cyanobacteria. This pigment absorbs UV radiation protecting subtending cyanobacterial cells from harmful effects. Based on scytonemin’s unique chemical structure, the pathway to its biosynthesis is uncertain, thus motivating the current investigation. Herein, we report the incorporation of both tyrosine and tryptophan into scytonemin, and provide in vivo data supporting the tryptophan origin of the ketone carbon involved in the condensation of the two biosynthetic precursors. This study also reports on the new use of a small-scale, MALDI-TOF mass spectrometry technique to monitor the incorporation of isotopically labeled tyrosine during scytonemin biosynthesis.