New additions to the arsenal of biocatalysts for noncanonical amino acid synthesis

Noncanonical amino acids (ncAAs) merge the conformational behavior and native interactions of proteinogenic amino acids with nonnative chemical motifs and have proven invaluable in developing modern therapeutics. This blending of native and nonnative characteristics has resulted in essential drugs like nirmatrelvir, which comprises three ncAAs and is used to treat COVID-19. Enzymes are appearing prominently in recent syntheses of ncAAs, where they demonstrate impressive control over the stereocenters and functional groups found therein. Here we review recent efforts to expand the biocatalyst arsenal for synthesizing ncAAs with natural enzymes. We also discuss how new-to-nature enzymes can contribute to this effort by catalyzing reactions inspired by the vast repertoire of chemical catalysis and acting on substrates that would otherwise not be used in synthesizing ncAAs. Abiotic enzyme-catalyzed reactions exploit the selectivity afforded by a macromolecular catalyst to access molecules not available to natural enzymes and perhaps not even chemical catalysis.     

Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.     

Initiation of Protein Synthesis with Non-Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.     

Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains

The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein–protein interactions, and improve the pharmacological properties of proteins. We report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra‐ or intermolecular protein crosslinks. We evolved a pyrrolysyl‐tRNA synthetase that incorporates site‐specifically PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification. FPheK reacted with adjacent Lys, Cys, and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab.     

Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy

Super‐resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine‐based GCE system for click chemistry, combined with DNA‐PAINT microscopy, enables the visualization of even low‐abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue‐specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.     

Incorporation of Non‐canonical Amino Acids into 2,5‐Diketopiperazines by Cyclodipeptide Synthases

The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5‐diketopiperazines (2,5‐DKPs) have been widely developed by medicinal chemists, but their biological production is yet to be exploited. We introduce an in vivo method for incorporating non‐canonical amino acids (ncAAs) into 2,5‐DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5‐DKP biosynthetic pathways. CDPSs use aminoacyl‐tRNAs as substrates. We exploited the natural ability of aminoacyl‐tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non‐canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5‐DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide‐tailoring enzymes found in 2,5‐DKP biosynthetic pathways.     

Selection of horseradish peroxidase variants with enhanced enantioselectivity by yeast surface display

We report a method for in vitro selection of catalytically active enzymes from large libraries of variants displayed on the surface of the yeast S. cerevisiae. Two libraries, each containing approximately 2 x 10(6) variants of horseradish peroxidase (HRP), were constructed; one involved error-prone PCR that sampled mutations throughout the coding sequence, whereas the other involved complete combinatorial enumeration of five positions near the active site to non-cysteine residues. The enzyme variants displayed on the yeast surface were allowed to modify it with a fluorescently labeled substrate. A combination of positive and negative selection applied to the active-site-directed library resulted in variants with up to an 8-fold altered enantioselectivity, including its reversal, toward L/D-tyrosinol. In contrast, the library constructed by using error-prone PCR yielded no HRP variants with a significantly improved enantioselectivity.     

An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells**

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.     

Combinatorial Library Based Engineering of Candida antarctica Lipase A for Enantioselective Transacylation of sec‐Alcohols in Organic Solvent

A method for determining lipase enantioselectivity in the transacylation of sec‐alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1‐phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel‐coated 96‐well microtiter plates through a histidine tag (His₆‐tag), screened for transacylation of 1‐phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild‐type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.     

Regiodivergent and Enantioselective Hydroxylation of C−H bonds by Synergistic Use of Protein Engineering and Exogenous Dual-Functional Small Molecules

It is a great challenge to optionally access diverse hydroxylation products from a given substrate bearing multiple reaction sites of sp³ and sp² C−H bonds. Herein, we report the highly selective divergent hydroxylation of alkylbenzenes by an engineered P450 peroxygenase driven by a dual-functional small molecule (DFSM). Using combinations of various P450BM3 variants with DFSMs enabled access to more than half of all possible hydroxylated products from each substrate with excellent regioselectivity (up to >99 %), enantioselectivity (up to >99 % ee), and high total turnover numbers (up to 80963). Crystal structure analysis, molecular dynamic simulations, and theoretical calculations revealed that synergistic effects between exogenous DFSMs and the protein environment controlled regio- and enantioselectivity. This work has implications for exogenous-molecule-modulated enzymatic regiodivergent and enantioselective hydroxylation with potential applications in synthetic chemistry.     

A comparative study of the asymmetric epoxidation of aromatic olefins using the first generation manganese salen epoxidation catalysts and their light fluorous variants: an interesting discovery on the use of benzotrifluoride as a cosolvent

The asymmetric epoxidation of aromatic olefins using optically active first generation manganese salen catalysts and light fluorous variants was examined. Although a slight decrease in the enantioselectivity of the product was observed when light fluorous catalysts were employed, the activities of these catalysts were higher than those of the non-fluorous catalysts. Additionally the influence on enantioselectivity of the oxidation was examined when fluorous cosolvents were used. The enantioselectivity of the oxidation increased with the addition of benzotrifluoride (BTF) regardless of whether a fluorous or non-fluorous salen catalyst was used.     

Selecting Better Biocatalysts by Complementing Recoded Bacteria**

In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild-type enzyme for ncAA-precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts.     

Microseparation techniques for the study of the enantioselectivity of drug–plasma protein binding

Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. The investigation of enantioselectivity of drugs in their binding with human plasma proteins and the identification of the molecular mechanisms involved in the stereodiscrimination by the proteins represent a great challenge for clinical pharmacology. In this review, the separation techniques used for enantioselective protein binding experiments are described and compared. An overview of studies on enantiomer–protein interactions, enantiomer–enantiomer interactions as well as chiral drug–drug interactions, including allosteric effects, is presented. The contribution of individual plasma proteins to the overall enantioselective binding and the animal species variability in drug–plasma protein binding stereoselectivity are reviewed. Copyright © 2008 John Wiley & Sons, Ltd.     

Phenotyping of bovine milk proteins by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the separation of the most common and some less common genetic variants of the bovine caseins is described. When the method is used for analysing clarified skim milk, simultaneous identification of casein variants and major they protein variants can be effected in a single run. The potential of the method for quantitative application is discussed.     

Chemoenzymatic Cascade Synthesis of Optically Pure Alkanoic Acids by Using Engineered Arylmalonate Decarboxylase Variants

Arylmalonate decarboxylase (AMDase) catalyzes the cofactor-free asymmetric decarboxylation of prochiral arylmalonic acids and produces the corresponding monoacids with rigorous R selectivity. Alteration of catalytic cysteine residues and of the hydrophobic environment in the active site by protein engineering has previously resulted in the generation of variants with opposite enantioselectivity and improved catalytic performance. The substrate spectrum of AMDase allows it to catalyze the asymmetric decarboxylation of small methylvinylmalonic acid derivatives, implying the possibility to produce short-chain 2-methylalkanoic acids with high optical purity after reduction of the nonactivated C=C double bond. Use of diimide as the reductant proved to be a simple strategy to avoid racemization of the stereocenter during reduction. The developed chemoenzymatic sequential cascade with use of R- and S-selective AMDase variants produced optically pure short-chain 2-methylalkanoic acids in moderate to full conversion and gave both enantiomers in excellent enantiopurity (up to 83 % isolated yield and 98 % ee).     

Directed evolution of streptavidin variants using in vitro compartmentalization

We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities ( approximately 10(-13) M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25 degrees C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples.     

Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to β-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo β-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.     

Biocatalytic Synthesis of Allylic and Allenyl Sulfides through a Myoglobin‐Catalyzed Doyle–Kirmse Reaction

The first example of a biocatalytic [2,3]‐sigmatropic rearrangement reaction involving allylic sulfides and diazo reagents (Doyle–Kirmse reaction) is reported. Engineered variants of sperm whale myoglobin catalyze this synthetically valuable C?C bond‐forming transformation with high efficiency and product conversions across a variety of sulfide substrates (e.g., aryl‐, benzyl‐, and alkyl‐substituted allylic sulfides) and α‐diazo esters. Moreover, the scope of this myoglobin‐mediated transformation could be extended to the conversion of propargylic sulfides to give substituted allenes. Active‐site mutations proved effective in enhancing the catalytic efficiency of the hemoprotein in these reactions as well as modulating the enantioselectivity, resulting in the identification of the myoglobin variant Mb(L29S,H64V,V68F), which is capable of mediating asymmetric Doyle–Kirmse reactions with an enantiomeric excess up to 71 %. This work extends the toolbox of currently available biocatalytic strategies for the asymmetric formation of carbon–carbon bonds.     

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of （ R, S ） -2-Octanol Acetate

To identify the desired hyperthermophilic variants within a mutant esterase library for the resolution of (R,S)-2-octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96.2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.     

Time-dependent expression and processing of a hypothetical protein of possible importance for regulation of the Chlamydia pneumoniae developmental cycle.

Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract. It is a Gram-negative bacteria and has a unique biphasic developmental cycle. In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C. pneumoniae proteins. We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.