Transaminase-Catalyzed Synthesis of ß-Branched Noncanonical Amino Acids Driven by a Lysine Amine Donor.

Transaminases are choice biocatalysts for the synthesis of chiral primary amines, including amino acids bearing contiguous stereocenters. In this study, we employ lysine as a "smart" amine donor in transaminase-catalyzed dynamic kinetic resolution reactions to access ß-branched noncanonical arylalanines. Our mechanistic investigation demonstrates that, upon transamination, the lysine-derived ketone byproduct readily cyclizes to a six-membered imine, driving the equilibrium in the desired direction and thus alleviating the need to load superstoichiometric quantities of the amine donor or deploy a multienzyme cascade. Lysine also shows good overall compatibility with a panel of wild-type transaminases, a promising hint of its application as a smart donor more broadly. Indeed, by this approach, we furnished a broad scope of ß-branched arylalanines, including some bearing hitherto intractable cyclopropyl and isopropyl substituents, with high yields and excellent selectivities.

Asymmetric Alkylation of Ketones Catalyzed by Engineered TrpB.

The ß-subunit of tryptophan synthase (TrpB) catalyzes a PLP-mediated ß-substitution reaction between indole and serine to form L-Trp. A succession of TrpB protein engineering campaigns to expand the enzyme's nucleophile substrate range has enabled the biocatalytic production of diverse non-canonical amino acids (ncAAs). Here, we show that ketone-derived enolates can serve as nucleophiles in the TrpB reaction to achieve the asymmetric alkylation of ketones, an outstanding challenge in synthetic chemistry. We engineered TrpB by directed evolution to catalyze the asymmetric alkylation of propiophenone and 2-fluoroacetophenone with a high degree of selectivity. In reactions with propiophenone, preference for the opposite product diastereomer emerges over the course of evolution, demonstrating that full control over the stereochemistry at the new chiral center can be achieved. The addition of this new reaction to the TrpB platform is a crucial first step toward the development of efficient methods to synthesize non-canonical prolines and other chirally dense nitrogen heterocycles.

Nature's Machinery, Repurposed: Expanding the Repertoire of Iron-Dependent Oxygenases.

Iron is an especially important redox-active cofactor in biology because of its ability to mediate reactions with atmospheric O2. Iron-dependent oxygenases exploit this earth-abundant transition metal for the insertion of oxygen atoms into organic compounds. Throughout the astounding diversity of transformations catalyzed by these enzymes, the protein framework directs reactive intermediates toward the precise formation of products, which, in many cases, necessitates the cleavage of strong C-H bonds. In recent years, members of several iron-dependent oxygenase families have been engineered for new-to-nature transformations that offer advantages over conventional synthetic methods. In this Perspective, we first explore what is known about the reactivity of heme-dependent cytochrome P450 oxygenases and nonheme iron-dependent oxygenases bearing the 2-His-1-carboxylate facial triad by reviewing mechanistic studies with an emphasis on how the protein scaffold maximizes the catalytic potential of the iron-heme and iron cofactors. We then review how these cofactors have been repurposed for abiological transformations by engineering the protein frameworks of these enzymes. Finally, we discuss contemporary challenges associated with engineering these platforms and comment on their roles in biocatalysis moving forward.

Hydrogen Donation but not Abstraction by a Tyrosine (Y68) during Endoperoxide Installation by Verruculogen Synthase (FtmOx1).

Hydrogen-atom transfer (HAT) from a substrate carbon to an iron(IV)-oxo (ferryl) intermediate initiates a diverse array of enzymatic transformations. For outcomes other than hydroxylation, coupling of the resultant carbon radical and hydroxo ligand (oxygen rebound) must generally be averted. A recent study of FtmOx1, a fungal iron(II)- and 2-(oxo)glutarate-dependent oxygenase that installs the endoperoxide of verruculogen by adding O2 between carbons 21 and 27 of fumitremorgin B, posited that tyrosine (Tyr or Y) 224 serves as HAT intermediary to separate the C21 radical (C21•) and Fe(III)-OH HAT products and prevent rebound. Our reinvestigation of the FtmOx1 mechanism revealed, instead, direct HAT from C21 to the ferryl complex and surprisingly competitive rebound. The C21-hydroxylated (rebound) product, which undergoes deprenylation, predominates when low [O2] slows C21•-O2 coupling in the next step of the endoperoxidation pathway. This pathway culminates with addition of the C21-O-O• peroxyl adduct to olefinic C27 followed by HAT to the C26• from a Tyr. The last step results in sequential accumulation of Tyr radicals, which are suppressed without detriment to turnover by inclusion of the reductant, ascorbate. Replacement of each of four candidates for the proximal C26 H• donor (including Y224) with phenylalanine (F) revealed that only the Y68F variant (i) fails to accumulate the first Tyr• and (ii) makes an altered major product, identifying Y68 as the donor. The implied proximities of C21 to the iron cofactor and C26 to Y68 support a new docking model of the enzyme-substrate complex that is consistent with all available data.

α-Amine Desaturation of d-Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent l-Arginine 3-Hydroxylase, VioC.

When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an l-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, d-Arg. The reactant complex with d-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H•) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with NaB2H4 to produce racemic [2H]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of 18O from 18O2. The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the l-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.

Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of α-Heteroatom Assistance.

Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (2H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors 1H or 2H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors 2H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.

Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase.

Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C-H and ferryl Fe-O bonds, primarily by direction of the oxo group into one of two cis-related coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution X-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the l-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins.

The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.

The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a "channel dye" into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation-π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation-π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation-π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors.

Structure-Guided Reprogramming of a Hydroxylase To Halogenate Its Small Molecule Substrate.

Enzymatic installation of chlorine/bromine into unactivated carbon centers provides a versatile, selective, and environmentally friendly alternative to chemical halogenation. Iron(II) and 2-(oxo)-glutarate (FeII/2OG)-dependent halogenases are powerful biocatalysts that are capable of cleaving aliphatic C-H bonds to introduce useful functional groups, including halogens. Using the structure of the Fe/2OG halogenase, WelO5, in complex with its small molecule substrate, we identified a similar N-acyl amino acid hydroxylase, SadA, and reprogrammed it to halogenate its substrate, thereby generating a new chiral haloalkyl center. The work highlights the potential of FeII/2OG enzymes as platforms for development of novel stereospecific catalysts for late-stage C-H functionalization.