RESUMEN
BACKGROUND: Next-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours. When compared to the traditional ligation-based methods, transposed-based Nextera has a slight insertion bias. RESULTS: Here we present the discovery of a mutant transposase (Tn5-059) with a lowered GC insertion bias through protein engineering. We demonstrate Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. We also observe higher library diversity generated by Tn5-059 when compared to Nextera v2 for human exomes, which leads to less sequencing and lower cost per genome. In addition, when used for human exomes, Tn5-059 delivers consistent library insert size over a range of input DNA, allowing up to a tenfold variance from the 50 ng input recommendation. CONCLUSIONS: Enhanced DNA input tolerance of Tn5-059 can translate to flexibility and robustness of workflow. DNA input tolerance together with superior uniformity of coverage and lower AT dropouts extend the applications of transposase based library preps. We discuss possible mechanisms of improvements in Tn5-059, and potential advantages of using the new mutant in varieties of applications including microbiome sequencing and chromatin profiling.
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Mapeo Cromosómico/métodos , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ingeniería de Proteínas , Análisis de Secuencia de ADN/métodos , Transposasas/genética , Secuencia Rica en At/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.
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ADN Polimerasa Dirigida por ADN/química , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nucleótidos/genética , Análisis de Secuencia de ADN , Bacteriófago phi X 174/genética , Disparidad de Par Base , Secuencia de Bases , ADN/química , Diseño de Equipo , Genoma Viral , Genómica , Cinética , Datos de Secuencia Molecular , PolímerosRESUMEN
The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.
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Oxidorreductasas de Alcohol/química , Aldehído Oxidorreductasas/química , Lignina/biosíntesis , Medicago truncatula/enzimología , Petunia/enzimología , Propanoles/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Aldehído Oxidorreductasas/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Cisteína/metabolismo , Disulfuros/metabolismo , Ésteres/metabolismo , Cinética , Ligandos , Lignina/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADP/metabolismo , Propanoles/química , Homología Estructural de Proteína , Especificidad por Sustrato , TemperaturaRESUMEN
The cephalochordate Amphioxus naturally co-expresses fluorescent proteins (FPs) with different brightness, which thus offers the rare opportunity to identify FP molecular feature/s that are associated with greater/lower intensity of fluorescence. Here, we describe the spectral and structural characteristics of green FP (bfloGFPa1) with perfect (100%) quantum efficiency yielding to unprecedentedly-high brightness, and compare them to those of co-expressed bfloGFPc1 showing extremely-dim brightness due to low (0.1%) quantum efficiency. This direct comparison of structure-function relationship indicated that in the bright bfloGFPa1, a Tyrosine (Tyr159) promotes a ring flipping of a Tryptophan (Trp157) that in turn allows a cis-trans transformation of a Proline (Pro55). Consequently, the FP chromophore is pushed up, which comes with a slight tilt and increased stability. FPs are continuously engineered for improved biochemical and/or photonic properties, and this study provides new insight to the challenge of establishing a clear mechanistic understanding between chromophore structural environment and brightness level.
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Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Anfioxos/metabolismo , Pliegue de Proteína , Animales , Cristalización , Cristalografía por Rayos X , Proteínas Fluorescentes Verdes/clasificación , Modelos MolecularesRESUMEN
BACKGROUND: Green fluorescent protein (GFP) has been found in a wide range of Cnidaria, a basal group of metazoans in which it is associated with pigmentation, fluorescence, and light absorbance. A GFP has been recently discovered in the pigmentless chordate Branchiostoma floridae (amphioxus) that shows intense fluorescence mainly in the head region. RESULTS: The amphioxus genome encodes 16 closely-related GFP-like proteins, all of which appear to be under purifying selection. We divide them into 6 clades based on protein sequence identity and show that representatives of each clade have significant differences in fluorescence intensity, extinction coefficients, and absorption profiles. Furthermore, GFPs from two clades exhibit antioxidant capacity. We therefore propose that amphioxus GFPs have diversified their functions into fluorescence, redox, and perhaps just light absorption in relation to pigmentation and/or photoprotection. CONCLUSION: The rapid radiation of amphioxus GFP into clades with distinct functions and spectral properties reveals functional plasticity of the GFP core. The high sequence similarities between different clades provide a model system to map sequence variation to functional changes, to better understand and engineer GFP.
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Cordados no Vertebrados/genética , Evolución Molecular , Proteínas Fluorescentes Verdes/genética , Familia de Multigenes , Animales , Antioxidantes/metabolismo , Proteínas Fluorescentes Verdes/aislamiento & purificación , Proteínas Fluorescentes Verdes/metabolismo , Filogenia , Análisis de Secuencia de Proteína , Espectrometría de FluorescenciaRESUMEN
4,2',4',6'-Tetrahydroxychalcone (chalcone) and 4,2',4'-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/keto-reductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase.
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Oxidorreductasas de Alcohol/química , Chalcona/análogos & derivados , Sitios de Unión , Chalcona/química , Chalcona/metabolismo , Chalconas , Cromatografía en Gel , Cristalografía por Rayos X , Ésteres/metabolismo , Evolución Molecular , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Medicago sativa/enzimología , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas de Alcohol Dependientes de NAD (+) y NADP (+) , NADP/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Plants, as sessile organisms, evolve and exploit metabolic systems to create a rich repertoire of complex natural products that hold adaptive significance for their survival in challenging ecological niches on earth. As an experimental tool set, structural biology provides a high-resolution means to uncover detailed information about the structure-function relationships of metabolic enzymes at the atomic level. Together with genomic and biochemical approaches and an appreciation of molecular evolution, structural enzymology holds great promise for addressing a number of questions relating to secondary or, more appropriately, specialized metabolism. Why is secondary metabolism so adaptable? How are reactivity, regio-chemistry and stereo-chemistry steered during the multi-step conversion of substrates into products? What are the vestigial structural and mechanistic traits that remain in biosynthetic enzymes during the diversification of substrate and product selectivity? What does the catalytic landscape look like as an enzyme family traverses all possible lineages en route to the acquisition of new substrate and/or product specificities? And how can one rationally engineer biosynthesis using the unique perspectives of evolution and structural biology to create novel chemicals for human use?
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Fenilalanina/análogos & derivados , Plantas/enzimología , Plantas/metabolismo , Evolución Biológica , Fenilalanina/biosíntesis , Plantas/genética , Relación Estructura-ActividadRESUMEN
We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.