RESUMEN
Biocatalysis, using enzymes for organic synthesis, has emerged as powerful tool for the synthesis of active pharmaceutical ingredients (APIs). The first industrial biocatalytic processes launched in the first half of the last century exploited whole-cell microorganisms where the specific enzyme at work was not known. In the meantime, novel molecular biology methods, such as efficient gene sequencing and synthesis, triggered breakthroughs in directed evolution for the rapid development of process-stable enzymes with broad substrate scope and good selectivities tailored for specific substrates. To date, enzymes are employed to enable shorter, more efficient, and more sustainable alternative routes toward (established) small molecule APIs, and are additionally used to perform standard reactions in API synthesis more efficiently. Herein, large-scale synthetic routes containing biocatalytic key steps toward >130 APIs of approved drugs and drug candidates are compared with the corresponding chemical protocols (if available) regarding the steps, reaction conditions, and scale. The review is structured according to the functional group formed in the reaction.
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Biocatálisis , Preparaciones FarmacéuticasRESUMEN
Enzymes and peptide catalysts consist of the same building blocks but require vastly different environments to operate best. Herein, we show that an enzyme and a peptide catalyst can work together in a single reaction vessel to catalyze a two-step cascade reaction with high chemo- and stereoselectivity. Abundant linear alcohols, nitroolefins, an alcohol oxidase, and a tripeptide catalyst provided chiral γ-nitroaldehydes in aqueous buffer. High yields (up to 92 %) and stereoselectivities (up to 98 % ee) were achieved for the cascade through the rational design of the peptide catalyst and the identification of common reaction conditions.
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Alcoholes , Péptidos , Estereoisomerismo , CatálisisRESUMEN
Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1-50â mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.
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Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas/métodos , Cromatografía Líquida de Alta Presión/métodos , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi-enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3-amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)- and (S)-selective TAs, both diastereomers of the 1,3-amino alcohol motif were accessible. A two-step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products.
RESUMEN
We report on novel chemoenzymatic routes toward tenofovir using low-cost starting materials and commercial or homemade enzyme preparations as biocatalysts. The biocatalytic key step was accomplished either via stereoselective reduction using an alcohol dehydrogenase or via kinetic resolution using a lipase. By employing a suspension of immobilized lipase from Burkholderia cepacia (Amano PS-IM) in a mixture of vinyl acetate and toluene, the desired (R)-ester (99% ee) was obtained on a 500 mg scale (60 mM) in 47% yield. Alternatively, stereoselective reduction of 1-(6-chloro-9H-purin-9-yl) propan-2-one (84 mg, 100 mM) catalyzed by lyophilized E. coli cells harboring recombinant alcohol dehydrogenase (ADH) from Lactobacillus kefir (E. coli/Lk-ADH Prince) allowed one to reach quantitative conversion, 86% yield and excellent optical purity (>99% ee) of the corresponding (R)-alcohol. The key (R)-intermediate was transformed into tenofovir through "one-pot" aminolysis-hydrolysis of (R)-acetate in NH3-saturated methanol, alkylation of the resulting (R)-alcohol with tosylated diethyl(hydroxymethyl) phosphonate, and bromotrimethylsilane (TMSBr)-mediated cleavage of the formed phosphonate ester into the free phosphonic acid. The elaborated enzymatic strategy could be applicable in the asymmetric synthesis of tenofovir prodrug derivatives, including 5'-disoproxil fumarate (TDF, Viread) and 5'-alafenamide (TAF, Vemlidy). The molecular basis of the stereoselectivity of the employed ADHs was revealed by molecular docking studies.
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Fármacos Anti-VIH , Organofosfonatos , Tenofovir , Alcohol Deshidrogenasa , Escherichia coli , Simulación del Acoplamiento Molecular , Alanina , LipasaRESUMEN
Identifying (bio)catalysts displaying high enantio-/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, a high-throughput, stereoselective screening assay is reported that gives information on enantioselectivity, stereopreference and activity as showcased for peroxygenase-catalyzed hydroxylation. The assay is based on spectrophotometric analysis of the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec-alcohol product formed in the peroxygenase reaction. The assay was applied to investigate a library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not reported yet. Thereby, previously non-described wild-type UPOs displaying (S)- as well as (R)-stereoselectivity for the hydroxylation of representative model substrates were identified, reaching up to 98 % ee for the (R)- and 94 % ee for the (S)-enantiomer. Homology models with concomitant docking studies indicated the structural reason for the observed complementary stereopreference.
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Oxigenasas de Función Mixta , Estereoisomerismo , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , CatálisisRESUMEN
Regioselective carbon-carbon bond formation belongs to the challenging tasks in organic synthesis. In this context, C-C bond formation catalyzed by 4-dimethylallyltryptophan synthases (4-DMATSs) represents a possible tool to regioselectively synthesize C4-prenylated indole derivatives without site-specific preactivation and circumventing the need of protection groups as used in chemical synthetic approaches. In this study, a toolbox of 4-DMATSs to produce a set of 4-dimethylallyl tryptophan and indole derivatives was identified. Using three wild-type enzymes as well as variants, various C5-substituted tryptophan derivatives as well as N-methyl tryptophan were successfully prenylated with conversions up to 90 %. Even truncated tryptophan derivatives like tryptamine and 3-indole propanoic acid were regioselectively prenylated in position C4. The acceptance of C5-substituted tryptophan derivatives was improved up to 5-fold by generating variants (e. g. T108S). The feasibility of semi-preparative prenylation of selected tryptophan derivatives was successfully demonstrated on 100â mg scale at 15â mM substrate concentration, allowing to reduce the previously published multistep chemical synthetic sequence to just a single step.
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Dimetilaliltranstransferasa , Triptófano , Biocatálisis , Carbono , Dimetilaliltranstransferasa/metabolismo , Indoles/química , Prenilación , Especificidad por Sustrato , Triptófano/metabolismoRESUMEN
Alcohol dehydrogenases (ADHs; EC 1.1.1.1) have been widely used for the reversible redox reactions of carbonyl compounds (i.e., aldehydes and ketones) and primary or secondary alcohols, often resulting in optically pure hydroxyl products with high added value. In this work, we report a concise chemoenzymatic route toward xanthine-based enantiomerically pure active pharmaceutical ingredients (API) - proxyphylline, xanthinol, and diprophylline employing various recombinant short-chain ADHs with (R)- or (S)-selectivity as key biocatalysts. By choosing the appropriate ADH, the (R)- as well as the (S)-enantiomer of proxyphylline was prepared in excellent enantiomeric excess (99-99.9% ee), >99% conversion, and the isolated yield ranging from 65% to 74%, depending on the used biocatalyst (ADH-A from Rhodococcus ruber or a variant derived from Lactobacillus kefir, Lk-ADH-Lica). In turn, E. coli/ADH-catalyzed bioreduction of the carbonylic precursor of xanthinol and diprophylline furnished the corresponding (S)-chlorohydrin in >99% ee, >99% conversion, and 80% yield (in the case of Lk-ADH-Lica); while the (R)-counterpart was afforded in 94% ee, 64% conversion, and 41% yield (in the case of SyADH from Sphingobium yanoikuyae). After further chemical functionalization of the key (S)-chlorohydrin intermediate, the desired homochiral (R)-xanthinol (>99% ee) was obtained in 97% yield and (S)-diprophylline (>99% ee) in 90% yield. The devised biocatalytic method is straightforward and thus might be considered practical in the manufacturing of title pharmaceuticals.
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Clorhidrinas , Difilina , Biocatálisis , Escherichia coli , Hidrógeno , Estereoisomerismo , Teofilina/análogos & derivadosRESUMEN
Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.
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Enterocolitis Seudomembranosa/genética , Enterotoxinas/metabolismo , Interacciones Microbiota-Huesped/genética , Klebsiella oxytoca/genética , Animales , Benzodiazepinonas/metabolismo , Benzodiazepinonas/toxicidad , Daño del ADN/efectos de los fármacos , Enterocolitis Seudomembranosa/microbiología , Enterocolitis Seudomembranosa/patología , Enterotoxinas/biosíntesis , Células Epiteliales/microbiología , Células Epiteliales/patología , Humanos , Intestinos/microbiología , Intestinos/patología , Infecciones por Klebsiella/genética , Infecciones por Klebsiella/microbiología , Klebsiella oxytoca/metabolismo , Klebsiella oxytoca/patogenicidad , Ratones , Microtúbulos/efectos de los fármacos , Oxiquinolina/análogos & derivados , Oxiquinolina/metabolismo , Oxiquinolina/toxicidad , Péptidos/metabolismo , Péptidos/toxicidadRESUMEN
The concurrent operation of chemical and biocatalytic reactions in one pot is still a challenging task, and, in particular for chemical photocatalysts, examples besides simple cofactor recycling systems are rare. However, especially due to the complementary chemistry that the two fields of catalysis promote, their combination in one pot has the potential to unlock intriguing, unprecedented overall reactivities. Herein we demonstrate a concurrent biocatalytic reduction and photocatalytic oxidation process. Specifically, the enantioselective biocatalytic sulfoxide reduction using (S)-selective methionine sulfoxide reductases was coupled to an unselective light-dependent sulfoxidation. Protochlorophyllide was established as a new green photocatalyst for the sulfoxidation. Overall, a cyclic deracemization process to produce nonracemic sulfoxides was achieved and the target compounds were obtained with excellent conversions (up to 91 %) and superb optical purity (>99 % ee).
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Sulfóxidos , Oxidación-Reducción , Sulfóxidos/químicaRESUMEN
Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.
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Cianobacterias , Electrones , Biocatálisis , Cianobacterias/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Agua/metabolismoRESUMEN
The substrate scope of the asymmetric allylation with zinc organyls catalyzed by 3,3-bis(2,4,6-triisopropylphenyl)-1,1-binaphthyl-2,2-diyl hydrogenphosphate (TRIP) has been extended to non-cyclic ester organozinc reagents and ketones. Tertiary chiral alcohols are obtained with ee's up to 94% and two stereogenic centers can be created. Compared to the previous lactone reagent the stereopreference switches almost completely, proving the fact that the nature of the organometallic compound is of immense importance for the asymmetry of the product.
RESUMEN
Demethylating methyl phenyl ethers is challenging, especially when the products are catechol derivatives prone to follow-up reactions. For biocatalytic demethylation, monooxygenases have previously been described requiring molecular oxygen which may cause oxidative side reactions. Here we show that such compounds can be demethylated anaerobically by using cobalamin-dependent methyltransferases exploiting thiols like ethyl 3-mercaptopropionate as a methyl trap. Using just two equivalents of this reagent, a broad spectrum of substituted guaiacol derivatives were demethylated, with conversions mostly above 90 %. This strategy was used to prepare the highly valuable antioxidant hydroxytyrosol on a one-gram scale in 97 % isolated yield.
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Guayacol/metabolismo , Oxigenasas de Función Mixta/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Biocatálisis , Desmetilación , Guayacol/química , Oxigenasas de Función Mixta/química , Estructura Molecular , Compuestos de Sulfhidrilo/químicaRESUMEN
Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).
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Acetofenonas/química , Alcohol Deshidrogenasa/química , Derivados del Benceno/química , Oxigenasas de Función Mixta/química , Nitrilos/química , Alcohol Feniletílico/química , Acetofenonas/metabolismo , Agrocybe/enzimología , Alcohol Deshidrogenasa/metabolismo , Derivados del Benceno/metabolismo , Catálisis , Luz , Oxigenasas de Función Mixta/metabolismo , Estructura Molecular , Nitrilos/metabolismo , Oxidación-Reducción , Alcohol Feniletílico/metabolismo , Procesos Fotoquímicos , Rhodococcus/enzimología , EstereoisomerismoRESUMEN
The Pictet-Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-ß-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR) catalyzes the Pictet-Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept short-chain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% ee STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila (OpSTR) using a combination of X-ray crystallography, mutational, and molecular dynamics (MD) studies. We discovered that short-chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.
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Liasas de Carbono-Nitrógeno/metabolismo , Catálisis , Unión Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
Stereoselective catalysts for the Pictet-Spengler reaction of tryptamines and aldehydes may allow a simple and fast approach to chiral 1-substituted tetrahydro-ß-carbolines. Although biocatalysts have previously been employed for the Pictet-Spengler reaction, not a single one accepts benzaldehyde and its substituted derivatives. To address this challenge, a combination of substrate walking and transfer of beneficial mutations between different wild-type backbones was used to develop a strictosidine synthase from Rauvolfia serpentina (RsSTR) into a suitable enzyme for the asymmetric Pictet-Spengler condensation of tryptamine and benzaldehyde derivatives. The double variant RsSTR V176L/V208A accepted various ortho-, meta- and para-substituted benzaldehydes and produced the corresponding chiral 1-aryl-tetrahydro-ß-carbolines with up to 99 % enantiomeric excess.
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Carbolinas , Caminata , Biocatálisis , Catálisis , EstereoisomerismoRESUMEN
3,3-Bis(2,4,6-triisopropylphenyl)-1,1-binaphthyl-2,2-diyl hydrogenphosphate (TRIP) catalyzes the asymmetric allylation of aldehydes with organozinc compounds, leading to highly valuable structural motifs, like precursors to lignan natural products. Our previously reported mechanistic proposal relies on two reaction intermediates and requires further investigation to really understand the mode of action and the origins of stereoselectivity. Detailed ab initio calculations, supported by experimental data, render a substantially different mode of action to the allyl boronate congener. Instead of a Brønsted acid-based catalytic activation, the chiral phosphate acts as a counterion for the Lewis acidic zinc ion, which provides the activation of the aldehyde.
RESUMEN
The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where all reaction steps are performed simultaneously, sequentially, or in flow.
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Enzimas/metabolismo , Compuestos Orgánicos/metabolismo , Bacterias/enzimología , Biocatálisis , Enzimas/química , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Lacasa/química , Lacasa/metabolismo , Metales/química , Compuestos Orgánicos/química , Oxidación-ReducciónRESUMEN
Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed fsqB (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme-like enzymes possess distinct structural properties, prompting us to investigate the structure-function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 Å resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. N-methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the meta-hydroxyl group, as e.g. in l-N-methyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product l-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the d-stereoisomer of N-methyltyrosine, in contrast to N-methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.
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Aspergillus fumigatus/enzimología , Dihidroxifenilalanina/metabolismo , Proteínas Fúngicas/química , Monoaminooxidasa/química , Aspergillus fumigatus/genética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Ciclización , Dihidroxifenilalanina/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Familia de Multigenes , Oxidación-Reducción , Especificidad por SustratoRESUMEN
C-C bond-forming reactions are key transformations for setting up the carbon frameworks of organic compounds. In this context, Friedel-Crafts acylation is commonly used for the synthesis of aryl ketones, which are common motifs in many fine chemicals and natural products. A bacterial multicomponent acyltransferase from Pseudomonas protegens (PpATase) catalyzes such Friedel-Crafts C-acylation of phenolic substrates in aqueous solution, reaching up to >99 % conversion without the need for CoA-activated reagents. We determined X-ray crystal structures of the native and ligand-bound complexes. This multimeric enzyme consists of three subunits: PhlA, PhlB, and PhlC, arranged in a Phl(A2 C2 )2 B4 composition. The structure of a reaction intermediate obtained from crystals soaked with the natural substrate 1-(2,4,6-trihydroxyphenyl)ethanone together with site-directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing a significant role during catalysis. These structural and mechanistic insights form the basis of further enzyme engineering efforts directed towards enhancing the substrate scope of this enzyme.