RESUMEN
Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include frontline antimicrobial and anticancer drugs such as erythromycin and doxorubicin. To replenish the clinicians' diminishing arsenal of bioactive molecules, a promising strategy aims at transferring polyketide biosynthetic pathways from their native producers into the biotechnologically desirable host Escherichia coli. This approach has been successful for type I modular polyketide synthases (PKSs); however, despite more than 3 decades of research, the large and important group of type II PKSs has until now been elusive in E. coli. Here, we report on a versatile polyketide biosynthesis pipeline, based on identification of E. coli-compatible type II PKSs. We successfully express 5 ketosynthase (KS) and chain length factor (CLF) pairs-e.g., from Photorhabdus luminescens TT01, Streptomyces resistomycificus, Streptoccocus sp. GMD2S, Pseudoalteromonas luteoviolacea, and Ktedonobacter racemifer-as soluble heterodimeric recombinant proteins in E. coli for the first time. We define the anthraquinone minimal PKS components and utilise this biosynthetic system to synthesise anthraquinones, dianthrones, and benzoisochromanequinones (BIQs). Furthermore, we demonstrate the tolerance and promiscuity of the anthraquinone heterologous biosynthetic pathway in E. coli to act as genetically applicable plug-and-play scaffold, showing it to function successfully when combined with enzymes from phylogenetically distant species, endophytic fungi and plants, which resulted in 2 new-to-nature compounds, neomedicamycin and neochaetomycin. This work enables plug-and-play combinatorial biosynthesis of aromatic polyketides using bacterial type II PKSs in E. coli, providing full access to its many advantages in terms of easy and fast genetic manipulation, accessibility for high-throughput robotics, and convenient biotechnological scale-up. Using the synthetic and systems biology toolbox, this plug-and-play biosynthetic platform can serve as an engine for the production of new and diversified bioactive polyketides in an automated, rapid, and versatile fashion.
Asunto(s)
Antraquinonas/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Proteínas Recombinantes/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/clasificación , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Antraquinonas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Vías Biosintéticas , Escherichia coli/genética , Modelos Químicos , Estructura Molecular , Filogenia , Hidrocarburos Policíclicos Aromáticos/química , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Policétidos/química , Proteínas Recombinantes/químicaRESUMEN
The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the α subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes.
Asunto(s)
Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Pseudomonas mendocina/enzimología , Sitios de Unión , Coenzimas/metabolismo , Cristalografía por Rayos X , Dimetilaminas/metabolismo , Mononucleótido de Flavina/metabolismo , Hemo/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , NADP/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Tetrahidrofolatos/metabolismoRESUMEN
Nature has evolved halogenase enzymes to regioselectively halogenate a diverse range of biosynthetic precursors, with the halogens introduced often having a profound effect on the biological activity of the resulting natural products. Synthetic endeavors to create non-natural bioactive small molecules for pharmaceutical and agrochemical applications have also arrived at a similar conclusion: halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo. Consequently, a high proportion of pharmaceuticals and agrochemicals on the market today possess halogens. Halogenated organic compounds are also common intermediates in synthesis and are particularly valuable in metal-catalyzed cross-coupling reactions. Despite the potential utility of organohalogens, traditional nonenzymatic halogenation chemistry utilizes deleterious reagents and often lacks regiocontrol. Reliable, facile, and cleaner methods for the regioselective halogenation of organic compounds are therefore essential in the development of economical and environmentally friendly industrial processes. A potential avenue toward such methods is the use of halogenase enzymes, responsible for the biosynthesis of halogenated natural products, as biocatalysts. This Review will discuss advances in developing halogenases for biocatalysis, potential untapped sources of such biocatalysts and how further optimization of these enzymes is required to achieve the goal of industrial scale biohalogenation.
Asunto(s)
Oxidorreductasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Catálisis , Halógenos/metabolismo , Oxidorreductasas/química , Peroxidasas/química , Peroxidasas/metabolismo , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Estereoisomerismo , Especificidad por Sustrato , Elementos de Transición/químicaRESUMEN
Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)-reticulene at which point the pathway diverges. Coclaurine N-methyltransferase (CNMT) is a key enzyme in the pathway to (S)-reticulene, installing the N-methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co-factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives.
Asunto(s)
Alcaloides/biosíntesis , Metiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Alcaloides/química , Bencilisoquinolinas/química , Bencilisoquinolinas/metabolismo , Biocatálisis , Dominio Catalítico , Coptis/enzimología , Cristalografía por Rayos X , Cinética , Metiltransferasas/química , Metiltransferasas/genética , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Especificidad por SustratoRESUMEN
Flavin-dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high-throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin "non-natural" product in E. coli.
RESUMEN
Flavin-dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X-ray crystal structure of a tryptophan 6-halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure-guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3-indolepropionate 75 % 5-chlorination was observed with the mutant in comparison to 90 % 6-chlorination for the wild-type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non-indolic substrates.
Asunto(s)
Oxidorreductasas/metabolismo , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Flavina-Adenina Dinucleótido/metabolismo , Halogenación , Indoles/química , Indoles/metabolismo , Cinética , Propionatos/química , Propionatos/metabolismo , Estructura Terciaria de Proteína , Estereoisomerismo , Especificidad por SustratoRESUMEN
Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.
Asunto(s)
FMN Reductasa/química , FMN Reductasa/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Dicroismo Circular , Cristalografía por Rayos X , Estabilidad de Enzimas , Cinética , Modelos Moleculares , Streptomyces/enzimología , Especificidad por Sustrato , Temperatura de TransiciónRESUMEN
Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to â¼10(12)-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5'-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys(629)-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems.
Asunto(s)
Proteínas Bacterianas/química , Clostridium sticklandii/química , Transferasas Intramoleculares/química , Fosfato de Piridoxal/química , Vitamina B 12/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Clostridium sticklandii/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Radicales Libres/química , Radicales Libres/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Cinética , Lisina/química , Lisina/metabolismo , Conformación Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ornitina/química , Ornitina/metabolismo , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Vitamina B 12/metabolismoRESUMEN
The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17=C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donor-acceptor distance for proton transfer.
Asunto(s)
Biocatálisis , Luz , Mutagénesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Biocatálisis/efectos de la radiación , Frío , Cianobacterias/enzimología , Cisteína , Cinética , Rayos Láser , Modelos Moleculares , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Conformación Proteica , Análisis EspectralRESUMEN
The light-activated enzyme NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the trans addition of hydrogen across the C-17-C-18 double bond of protochlorophyllide (Pchlide), a key step in chlorophyll biosynthesis. Similar to other members of the short chain alcohol dehydrogenase/reductase family of enzymes, POR contains a conserved Tyr and Lys residue in the enzyme active site, which are implicated in a proposed reaction mechanism involving proton transfer from the Tyr hydoxyl group to Pchlide. We have analyzed a number of POR variant enzymes altered in these conserved residues using a combination of steady-state turnover, laser photoexcitation studies, and low temperature fluorescence spectroscopy. None of the mutations completely abolished catalytic activity. We demonstrate their importance to catalysis by defining multiple roles in the overall reaction pathway. Mutation of either residue impairs formation of the ground state ternary enzyme-substrate complex, pointing to a key role in substrate binding. By analyzing the most active variant (Y193F), we show that Tyr-193 participates in proton transfer to Pchlide and stabilizes the Pchlide excited state, enabling hydride transfer from NADPH to Pchilde. Thus, in addition to confirming the probable identity of the proton donor in Pchlide reduction, our work defines additional roles for these residues in facilitating hydride transfer through stabilization of the ground and excited states of the ternary enzyme complex.
Asunto(s)
Frío , Rayos Láser , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Synechococcus/enzimología , Dominio Catalítico/genética , Fluorescencia , Cinética , Luz , Lisina/genética , Mutagénesis , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Procesos Fotoquímicos/efectos de la radiación , Estructura Terciaria de Proteína , Protones , Synechococcus/genética , Tirosina/genéticaRESUMEN
The light-driven enzyme, protochlorophyllide oxidoreductase (POR), has proven to be an excellent model system for studying the role of protein motions during catalysis. POR catalyzes the trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide (Pchlide), which is a key step in chlorophyll biosynthesis. While we currently have a detailed understanding of the initial photochemical events and the subsequent hydrogen transfer reactions, there remains a lack of information about the slower substrate binding events leading to the formation of the catalytically active ternary complex. As POR is light-activated, it is relatively straightforward to isolate the ternary enzyme-substrate complex in the dark prior to catalysis, which has facilitated the use of a variety of spectroscopic and kinetic probes to study the binding of both substrates. Herein, we provide a detailed kinetic and thermodynamic description of these processes and show that the binding events are complex, involving multiple conformational states en route to the formation of a ternary complex that is primed for photoactivation. The initial binding of NADPH involves three distinct steps, which appear to be necessary for the optimal alignment of the cofactor in the enzyme active site. This is followed by the binding of the Pchlide substrate and subsequent substrate-induced conformational changes within the enzyme that occur prior to the formation of the final "poised" conformational state. These studies, which provide important information on the formation of the reactive conformation, reveal that ternary complex formation is the rate-limiting step in the overall reaction and is controlled by slow conformational changes in the protein.
Asunto(s)
Luz , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Catálisis , Transferencia Resonante de Energía de Fluorescencia , Cinética , NADP/metabolismo , Unión Proteica , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Especificidad por SustratoRESUMEN
Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.
Asunto(s)
Luz , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Protoclorofilida/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADP/química , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Estructura Terciaria de Proteína , Protoclorofilida/química , Alineación de Secuencia , EspectrofotometríaRESUMEN
Despite major recent advances in C-H activation, discrimination between two similar, unactivated C-H positions is beyond the scope of current chemocatalytic methods. Here we demonstrate that integration of regioselective halogenase enzymes with Pd-catalysed cross-coupling chemistry, in one-pot reactions, successfully addresses this problem for the indole heterocycle. The resultant 'chemobio-transformation' delivers a range of functionally diverse arylated products that are impossible to access using separate enzymatic or chemocatalytic C-H activation, under mild, aqueous conditions. This use of different biocatalysts to select different C-H positions contrasts with the prevailing substrate-control approach to the area, and presents opportunities for new pathways in C-H activation chemistry. The issues of enzyme and transition metal compatibility are overcome through membrane compartmentalization, with the optimized process requiring no intermediate work-up or purification steps.
Asunto(s)
Oxidorreductasas/metabolismo , Catálisis , Dimetilpolisiloxanos/química , Halogenación , Ligandos , Paladio/metabolismo , EstereoisomerismoRESUMEN
How cobalamin-dependent enzymes promote C-Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5'-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C-Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12 -binding Rossmann domain to achieve a catalytically competent 'closed' conformational state. In 'closed' conformations of OAM, Glu338 is thought to facilitate C-Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C-Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C-Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5'-deoxyadenosyl group of B12 potentiate C-Co bond homolysis in 'closed' conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of 'closed' conformations of OAM. In 'closed' conformations, Glu338 interacts with the 5'-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C-Co homolysis, and is a crucial component of the approximately 10(12) rate enhancement achieved by cobalamin-dependent enzymes for C-Co bond homolysis.
Asunto(s)
Proteínas Bacterianas/química , Transferasas Intramoleculares/química , Sustitución de Aminoácidos , Biocatálisis , Clostridium sticklandii/enzimología , Ácido Glutámico/química , Cinética , Modelos Químicos , Ornitina/químicaRESUMEN
Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products.