RESUMEN
Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TE DAP ) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TE DAP , the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.
RESUMEN
A biocatalytic platform that employs the final two monomodular type I polyketide synthases of the pikromycin pathway in vitro followed by direct appendage of D-desosamine and final C-H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12- and 14-membered ring macrolide natural products. This methodology delivered both compound classes in 13 steps (longest linear sequence) from commercially available (R)-Roche ester in >10% overall yields.
Asunto(s)
Biocatálisis , Macrólidos/metabolismo , Biotransformación , Lactonas/metabolismo , Macrólidos/síntesis química , Sintasas Poliquetidas/metabolismoRESUMEN
The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926-1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ(1) but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ(1). Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.
Asunto(s)
Escherichia coli/enzimología , Evolución Molecular , Pentosiltransferasa/química , Secuencia de Aminoácidos , Guanina/análogos & derivados , Guanina/síntesis química , Guanina/química , Guanina/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Mutación , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Filogenia , Pirimidinonas/síntesis química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirroles/síntesis química , Pirroles/química , Pirroles/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The modified RNA base queuine [7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine] is present in tRNA because of a unique base-exchange process catalyzed by tRNA-guanine transglycosylase (TGT). Previous studies have suggested the intermediacy of a covalent TGT-RNA complex. To exist on the reaction pathway, this covalent complex must be both chemically and kinetically competent. Chemical competence has been demonstrated by the crystal structure studies of Xie et al. [(2003) Nat. Struct. Biol. 10, 781-788]; however, evidence of kinetic competence had not yet been established. The studies reported here unequivocally demonstrate that the TGT-RNA covalent complex is kinetically capable of occurring on the TGT reaction pathway. These studies further suggest that dissociation of product RNA from the enzyme is overall rate-limiting in the steady state. Interestingly, studies comparing RNA with a 2'-deoxyriboside at the site of modification suggest a role for the 2'-hydroxyl group in stabilizing the growing negative charge on the nucleophilic aspartate (264) as the glycosidic bond to the aspartate is broken during the breakdown of the covalent complex.
Asunto(s)
Escherichia coli/enzimología , Pentosiltransferasa/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Desoxirribosa/química , Desoxirribosa/metabolismo , Glicósidos/química , Glicósidos/metabolismo , Cinética , Modelos Químicos , Pentosiltransferasa/química , ARN/química , ARN/metabolismo , Especificidad por SustratoRESUMEN
The 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) polyketide synthase (PKS) are unique multifunctional enzyme systems that are responsible for the biosynthesis of the erythromycin and pikromycin 14-membered ring aglycones, respectively. Together, these natural product biosynthetic systems provide excellent platforms to examine the fundamental structural and catalytic elements that govern polyketide assembly, processing, and macrocyclization. In these studies, the native pentaketide intermediate for DEBS was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone products in engineered monomodules Ery5, Ery5-TE, and Ery6. A comparative analysis was performed with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), dissecting key similarities and differences between these highly related PKSs. The data revealed that individual modules in the DEBS and Pik PKSs possess distinctive molecular selectivity profiles and suggest that substrate recognition has evolved unique characteristics in each system.
Asunto(s)
Antibacterianos/metabolismo , Eritromicina/metabolismo , Macrólidos/metabolismo , Sintasas Poliquetidas/síntesis química , Cromatografía en Capa Delgada , Sintasas Poliquetidas/metabolismo , Especificidad por SustratoRESUMEN
The methymycin/pikromycin (Pik) macrolide pathway represents a robust metabolic system for analysis of modular polyketide biosynthesis. The enzymes that comprise this biosynthetic pathway display unprecedented substrate flexibility, combining to produce six structurally diverse macrolide antibiotics in Streptomyces venezuelae. Thus, it is appealing to consider that the pikromycin biosynthetic enzymes could be leveraged for high-throughput production of novel macrolide antibiotics. Accordingly, efforts over the past decade have focused on the detailed investigation of the six-module polyketide synthase, desosamine sugar assembly and glycosyl transfer, and the cytochrome P450 monooxygenase that is responsible for hydroxylation. This review summarizes the advances in understanding of pikromycin biosynthesis that have been gained during the course of these investigations.
Asunto(s)
Productos Biológicos/biosíntesis , Macrólidos/metabolismo , Productos Biológicos/química , Esterasas/metabolismo , Hidroxilación , Modelos Moleculares , Streptomyces/metabolismoRESUMEN
The pikromycin polyketide synthase (PKS) is unique in its ability to generate both 12 and 14 membered ring macrolactones. As such, dissection of the molecular basis for controlling metabolic diversity in this system remains an important objective for understanding modular PKS function and expanding chemical diversity. Here, we describe a series of experiments designed to probe the importance of the protein-protein interaction that occurs between the final two monomodules, PikAIII (module 5) and PikAIV (module 6), for the production of the 12 membered ring macrolactone 10-deoxymethynolide. The results obtained from these in vitro studies demonstrate that PikAIII and PikAIV generate the 12 membered ring macrocycle most efficiently when engaged in their native protein-protein interaction. Accordingly, the data are consistent with PikAIV adopting an alternative conformation that enables the terminal thioesterase domain to directly off-load the PikAIII-bound hexaketide intermediate for macrocyclization.
Asunto(s)
Lactonas/química , Macrólidos/metabolismo , Sintasas Poliquetidas/metabolismo , Secuencia de Bases , Dominio Catalítico , Ciclización , Cartilla de ADN , Esterasas/metabolismo , Mutagénesis Sitio-Dirigida , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genéticaRESUMEN
Within the large and diverse group of RNA-modifying enzymes, a number of enzymes seem to form stable covalent linkages to their respective RNA substrates. A complete understanding of the chemical and kinetic mechanisms of these enzymes, some of which have identified pathological roles, is lacking. As part of our ongoing work studying the posttranscriptional modification of tRNA with queuine, we wish to understand fully the chemical and kinetic mechanisms involved in this key transglycosylation reaction. In our previous investigations, we have used a gel mobility-shift assay to characterize an apparent covalent enzyme-RNA intermediate believed to be operative in the catalytic pathway. However, the simple observation of a covalent complex is not sufficient to prove intermediacy. To be a true intermediate, the complex must be both chemically and kinetically competent. As a case study for the proof of intermediacy, we report the use of this gel-shift assay under mildly denaturing conditions to probe the kinetic competency of the covalent association between RNA and the tRNA modifying enzyme tRNA-guanine transglycosylase (TGT).
Asunto(s)
Pentosiltransferasa/metabolismo , ARN/metabolismo , Animales , Humanos , Cinética , Pentosiltransferasa/química , ARN/químicaRESUMEN
Bacterial type I polyketide synthases (PKSs) are complex, multifunctional enzymes that synthesize structurally diverse and medicinally important natural products. Given their modular organization, the manipulation of type I PKSs holds tremendous promise for the generation of novel compounds that are not easily accessible by standard synthetic chemical approaches. In theory, hybrid polyketide synthetic pathways can be constructed through the rational recombination of catalytic domains or modules from a variety of PKS systems; however, the general success of this strategy has been elusive, largely due to a poor understanding of the interactions between catalytic domains, as well as PKS modules. Over the past several years, a fundamental knowledge of these issues, and others, has begun to emerge, offering refined strategies for the facile engineering of hybrid polyketide pathways.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Mejoramiento Genético/métodos , Macrólidos/metabolismo , Sintasas Poliquetidas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Complejos Multienzimáticos/metabolismoRESUMEN
Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.
Asunto(s)
Macrólidos/química , Tioléster Hidrolasas/química , Sitios de Unión/efectos de los fármacos , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Catálisis , Cristalización , Ciclización , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Macrólidos/metabolismo , Modelos Moleculares , Conformación Molecular , Organofosfonatos/química , Organofosfonatos/farmacología , Conformación Proteica , Estructura Terciaria de Proteína , Estereoisomerismo , Streptomyces/enzimología , Relación Estructura-Actividad , Tioléster Hidrolasas/antagonistas & inhibidores , Tioléster Hidrolasas/metabolismo , Difracción de Rayos XRESUMEN
Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.
Asunto(s)
Macrólidos/química , Tioléster Hidrolasas/química , Sitios de Unión , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Catálisis , Cristalización , Ciclización , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Macrólidos/metabolismo , Modelos Moleculares , Conformación Molecular , Organofosfonatos/química , Organofosfonatos/farmacología , Conformación Proteica , Estructura Terciaria de Proteína , Estereoisomerismo , Streptomyces/enzimología , Relación Estructura-Actividad , Tioléster Hidrolasas/antagonistas & inhibidores , Tioléster Hidrolasas/metabolismo , Difracción de Rayos XRESUMEN
The vast majority of the ca. 100 chemically distinct modified nucleosides in RNA appear to arise via the chemical transformation of a genetically encoded nucleoside. Two notable exceptions are queuosine and pseudouridine, which are incorporated into tRNA via transglycosylation. Transglycosylation is an extremely efficient process for incorporating highly modified bases such as queuine into RNA. Transglycosylation is also a requisite process for "isomerizing" an N-nucleoside into a C-nucleoside as is the case for pseudouridine formation. Finally, transglycosylation is an attractive possibility for certain RNA editing events (e.g., pyrimidine to purine conversions) that cannot occur via the known, more straightforward enzymatic reactions (e.g., deaminations). This review discusses what is known about the mechanisms of transglycosylation for the queuine and pseudouridine RNA modifications and will speculate about a potential role for transglycosylation in certain RNA editing events.
Asunto(s)
Edición de ARN , Procesamiento Postranscripcional del ARN/fisiología , ARN , Cristalografía por Rayos X , Glicosilación , Modelos Moleculares , Conformación Molecular , ARN/química , ARN/metabolismoRESUMEN
tRNA-guanine transglycosylase (TGT) catalyzes a post-transcriptional base-exchange reaction involved in the incorporation of the modified base queuine (Q) into the wobble position of certain tRNAs. Catalysis by TGT occurs through a double-displacement mechanism that involves the formation of a covalent enzyme-RNA intermediate (Kittendorf, J. D., Barcomb, L. M., Nonekowski, S. T., and Garcia, G. A. (2001) Biochemistry 40, 14123-14133). The TGT chemical mechanism requires the protonation of the displaced guanine and the deprotonation of the incoming heterocyclic base. Based on its position in the active site, it is likely that aspartate 264 is involved in these proton transfer events. To investigate this possibility, site-directed mutagenesis was employed to convert aspartate 264 to alanine, asparagine, glutamate, glutamine, lysine, and histidine. Biochemical characterization of these TGT mutants revealed that only the conservative glutamate mutant retained catalytic activity, with Km values for both tRNA and guanine 3-fold greater than those for wild-type, whereas the kcat was depressed by an order of magnitude. Furthermore, of these six TGT mutants, only the TGT(D264E) was capable of forming a TGT.RNA covalent intermediate; however, unlike wild-type TGT, only hydroxylamine is capable of cleaving the TGT(D264E).RNA covalent complex. In an effort to better understand the unique biochemical properties of the D264E TGT mutant, we solved the crystal structure of the Zymomonas mobilis TGT with the analogous mutation (D280E). The results of these studies support two roles for aspartate 264 in catalysis by TGT, protonation of the leaving guanine and deprotonation of the incoming preQ1.