Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.     

Cotranslational incorporation of a structurally diverse series of proline analogues in an Escherichia coli expression system

A set of Escherichia coli expression strains have been defined that are competent for the incorporation of a structurally diverse series of proline analogues under culture conditions that are compatible with high levels of analogue substitution within a proline-rich protein substrate. These bacterial strains have been employed to assay the efficacy of incorporation of noncanonical amino acids into a recombinant-protein test substrate and to create variant polypeptides in which native protein sequences have been globally substituted with imino acid analogues in response to proline codons. We envision that these methods may be used to interrogate the effect of imino acid substitution on protein structure and function and may be particularly informative in the context of structural comparison of a series of modified proteins with respect to the stereoelectronic differences between the incorporated proline analogues.     

Protein Macrocyclization for Tertiary Structure Stabilization

Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of proteins for biotechnological and biomedical purposes, it is often required to enforce their tertiary structure in order to ensure sufficient stability under the conditions inherent to the application of interest. The introduction of intramolecular crosslinks has proven efficient in stabilizing native protein folds. Herein, we give an overview of methods that allow the macrocyclization of expressed proteins, discussing involved reaction mechanisms and structural implications.     

Stereoselective incorporation of an unsaturated isoleucine analogue into a protein expressed in E. coli

The unsaturated amino acid 2-amino-3-methyl-4-pentenoic acid (E-Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS-E-Ile and SR-E-Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS-E-Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %; SR-E-Ile yielded no conclusive evidence for incorporation. ATP/PPi exchange assays indicated that SS-E-Ile was activated by the isoleucyl-tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR-E-Ile was activated approximately 100-times more slowly than SS-E-Ile.     

Phosphine-Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small-molecule triggers that turn on proteins through a Staudinger reduction/self-immolation cascade with substantially improved kinetics and yields. We achieved this through site-specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post-translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction-based activation, paving the way for its application in other proteins and organisms.     

B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase: Evaluation of the Proline Rule as a Method for Enzyme Stabilization

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. Here we present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60 °C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3 °C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the proline rule was deemed successful at increasing the stability of this enzyme.     

Tryptophan Synthase: Biocatalyst Extraordinaire

Tryptophan synthase (TrpS) has emerged as a paragon of noncanonical amino acid (ncAA) synthesis and is an ideal biocatalyst for synthetic and biological applications. TrpS catalyzes an irreversible, C−C bond-forming reaction between indole and serine to make l -tryptophan; native TrpS complexes possess fairly broad specificity for indole analogues, but are difficult to engineer to extend substrate scope or to confer other useful properties due to allosteric constraints and their heterodimeric structure. Directed evolution freed the catalytically relevant TrpS β-subunit (TrpB) from allosteric regulation by its TrpA partner and has enabled dramatic expansion of the enzyme's substrate scope. This review examines the long and storied career of TrpS from the perspective of its application in ncAA synthesis and biocatalytic cascades.     

Multiple Site‐Selective Insertions of Noncanonical Amino Acids into Sequence‐Repetitive Polypeptides

A simple and efficient method is described for the introduction of noncanonical amino acids at multiple, defined sites within recombinant polypeptide sequences. Escherichia coli MRA30, a bacterial host strain with attenuated activity of release factor 1 (RF1), was assessed for its ability to support incorporation of a diverse range of noncanonical amino acids in response to multiple encoded amber (TAG) codons within genes derived from superfolder GFP and an elastin‐mimetic protein polymer. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl‐tRNA synthetase/tRNA^CUA pair and the noncanonical amino acid. Elastin‐mimetic protein polymers were prepared in which noncanonical amino acid derivatives were incorporated at up to 22 specific sites within the polypeptide sequence with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full‐length polypeptides and proteolytic cleavage fragments from thermolysin digestion. The data suggest that this multisite suppression approach permits the preparation of protein‐based materials in which novel chemical functionalities can be introduced at precisely defined positions within the polypeptide sequence.     

Alteration of the Diastereoselectivity of 3‐Methylaspartate Ammonia Lyase by Using Structure‐Based Mutagenesis

3‐Methylaspartate ammonia‐lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)‐3‐methylaspartic acid and (2S,3R)‐3‐methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active‐site Mg^II ion. The crystal structure of MAL in complex with (2S,3S)‐3‐methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)‐3‐methylaspartic acid, and hence are likely candidates to assist the Mg^II ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site‐directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and ¹H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild‐type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of k_cat and k_cat/K_M; this implicates Lys331, His194 and Gln329 as mechanistically important. The ¹H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)‐3‐methylaspartic acid, with no detectable formation of (2S,3R)‐3‐methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate.