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1.
Mol Biol Int ; 2014: 287430, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25197572

RESUMEN

As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

2.
Front Microbiol ; 5: 474, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25250022

RESUMEN

Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus.

3.
Plant J ; 79(3): 517-29, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24905498

RESUMEN

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate-Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell-wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full-length coding sequences without termination codons and are Gateway® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/.


Asunto(s)
Genómica , Glicosiltransferasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Pared Celular/metabolismo
4.
PLoS One ; 9(6): e100836, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24971883

RESUMEN

Three lignocellulosic pretreatment techniques (ammonia fiber expansion, dilute acid and ionic liquid) are compared with respect to saccharification efficiency, particle size and biomass composition. In particular, the effects of switchgrass particle size (32-200) on each pretreatment regime are examined. Physical properties of untreated and pretreated samples are characterized using crystallinity, surface accessibility measurements and scanning electron microscopy (SEM) imaging. At every particle size tested, ionic liquid (IL) pretreatment results in greater cell wall disruption, reduced crystallinity, increased accessible surface area, and higher saccharification efficiencies compared with dilute acid and AFEX pretreatments. The advantages of using IL pretreatment are greatest at larger particle sizes (>75 µm).


Asunto(s)
Biomasa , Carbohidratos/biosíntesis , Pared Celular/efectos de los fármacos , Líquidos Iónicos/farmacología , Ácidos/farmacología , Pared Celular/ultraestructura , Cristalización , Microscopía Electrónica de Rastreo , Nitrógeno/química , Nitrógeno/metabolismo , Tamaño de la Partícula , Poaceae/química , Poaceae/metabolismo
5.
PLoS One ; 8(11): e79725, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24244549

RESUMEN

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.


Asunto(s)
Celulasa/metabolismo , Líquidos Iónicos , Poaceae , Thermotoga maritima/enzimología , Biomasa , Celulasa/química , Celulasa/genética , Activación Enzimática , Ensayos Analíticos de Alto Rendimiento , Modelos Moleculares , Mutagénesis , Conformación Proteica , Thermotoga maritima/genética
6.
Plant J ; 76(6): 1016-29, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24128328

RESUMEN

We have characterized a ß-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana that is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to the Carbohydrate Active Enzyme database glycosyltransferase family 14 (GT14). The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoris transferred glucuronic acid (GlcA) to ß-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to ß-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the ß-1,6- and ß-1,3-galactan present in type II AG. Two allelic T-DNA insertion mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild-type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-ß-1,6-Gal and ß-1,3-Gal, indicating an in vivo role of AtGlcAT14A in synthesis of those structures in type II AG. Moreover, a relative increase in the levels of 3-, 6- and 3,6-linked galactose (Gal) and reduced levels of 3-, 2- and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation in AtGlcAT14A results in a relative increase of the longer and branched ß-1,3- and ß-1,6-galactans. This increase of galactosylation in the mutants is most likely caused by increased availability of the O6 position of Gal, which is a shared acceptor site for AtGlcAT14A and galactosyltransferases in synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role for the glucuronosyltransferase in the biosynthesis of type II AG, with a biological role during seedling growth.


Asunto(s)
Arabidopsis/enzimología , Galactanos/biosíntesis , Glucuronosiltransferasa/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabinosa/genética , Arabinosa/metabolismo , Transporte Biológico , Dominio Catalítico , Pared Celular/metabolismo , Expresión Génica , Glucuronosiltransferasa/genética , Aparato de Golgi/metabolismo , Modelos Estructurales , Mutagénesis Insercional , Fenotipo , Filogenia , Pichia/enzimología , Pichia/genética , Proteínas Recombinantes , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Especificidad por Sustrato , Nicotiana/enzimología , Nicotiana/genética
7.
Front Microbiol ; 4: 282, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24069019

RESUMEN

Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26%) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase (GH) families were detected. Using tetranucleotide frequency (TNF) binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

8.
Biotechnol Biofuels ; 6(1): 93, 2013 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-23819686

RESUMEN

INTRODUCTION: Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. METHOD: We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. RESULTS: Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. CONCLUSION: Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

9.
BMC Biotechnol ; 12: 38, 2012 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-22759983

RESUMEN

BACKGROUND: Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. RESULTS: Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. CONCLUSIONS: Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.


Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Glicósido Hidrolasas/metabolismo , Metagenoma , Microbiología del Suelo , Bacterias/química , Bacterias/genética , Bacterias/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Sistemas de Lectura Abierta , Suelo
10.
Plant Physiol ; 159(4): 1408-17, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22706449

RESUMEN

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell walls that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of ß(1,4)-linked xylose residues substituted with α(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a K(m) for UDP-GlcA of 165 µm. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan α-glucuronosyltransferases.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Glucuronosiltransferasa/metabolismo , Glicosiltransferasas/metabolismo , Xilanos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Microsomas/enzimología , Modelos Moleculares , Filogenia , Transporte de Proteínas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/enzimología , Nicotiana/metabolismo
11.
Plant Physiol ; 159(1): 12-26, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22430844

RESUMEN

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.


Asunto(s)
Arabidopsis/metabolismo , Pared Celular/metabolismo , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteoma/aislamiento & purificación , Apirasa/genética , Apirasa/metabolismo , Arabidopsis/anatomía & histología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Centrifugación por Gradiente de Densidad , Cromatografía Liquida , Pruebas de Enzimas , Genes de Plantas , Prueba de Complementación Genética , Glicosilación , Aparato de Golgi/ultraestructura , Immunoblotting , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Células Vegetales/enzimología , Células Vegetales/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Proteómica/métodos , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
12.
Structure ; 19(12): 1876-84, 2011 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-22153510

RESUMEN

The sesquiterpene bisabolene was recently identified as a biosynthetic precursor to bisabolane, an advanced biofuel with physicochemical properties similar to those of D2 diesel. High-titer microbial bisabolene production was achieved using Abies grandis α-bisabolene synthase (AgBIS). Here, we report the structure of AgBIS, a three-domain plant sesquiterpene synthase, crystallized in its apo form and bound to five different inhibitors. Structural and biochemical characterization of the AgBIS terpene synthase Class I active site leads us to propose a catalytic mechanism for the cyclization of farnesyl diphosphate into bisabolene via a bisabolyl cation intermediate. Further, we describe the nonfunctional AgBIS Class II active site whose high similarity to bifunctional diterpene synthases makes it an important link in understanding terpene synthase evolution. Practically, the AgBIS crystal structure is important in future protein engineering efforts to increase the microbial production of bisabolene.


Asunto(s)
Abies/enzimología , Transferasas Alquil y Aril/química , Biocombustibles , Proteínas de Plantas/química , Transferasas Alquil y Aril/metabolismo , Dominio Catalítico , Proteínas de Plantas/metabolismo , Conformación Proteica , Sesquiterpenos/metabolismo
13.
Mol Syst Biol ; 7: 487, 2011 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-21556065

RESUMEN

Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n-butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes.


Asunto(s)
1-Butanol , Biocombustibles , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Proteínas de Transporte de Membrana/genética , Pentanoles , 1-Butanol/metabolismo , 1-Butanol/toxicidad , Biocombustibles/toxicidad , Biología Computacional , Escherichia coli/crecimiento & desarrollo , Proteínas de Transporte de Membrana/metabolismo , Redes y Vías Metabólicas , Análisis por Micromatrices , Pentanoles/metabolismo , Pentanoles/toxicidad
14.
Appl Microbiol Biotechnol ; 89(4): 989-1000, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20972784

RESUMEN

Biological synthesis of therapeutic drugs beneficial for human health using microbes offers an alternative production strategy to the methods that are commonly employed such as direct extraction from source organisms or chemical synthesis. In this study, we evaluated the potential for yeast (Saccharomyces cerevisiae) to be used as a catalyst for the synthesis of tranilast and various tranilast analogs (cinnamoyl anthranilates). Several studies have demonstrated that these phenolic amides have antioxidant properties and potential therapeutic benefits including antiinflammatory, antiproliferative, and antigenotoxic effects. The few cinnamoyl anthranilates naturally produced in plants such as oats and carnations result from the coupling of various hydroxycinnamoyl-CoAs to anthranilic acid. In order to achieve the microbial production of tranilast and several of its analogs, we engineered a yeast strain to co-express a 4-coumarate/CoA ligase (4CL, EC 6.2.1.12) from Arabidopsis thaliana and a hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, EC 2.3.1.144) from Dianthus caryophyllus. This modified yeast strain allowed us to produce tranilast and 26 different cinnamoyl anthranilate molecules within a few hours after exogenous supply of various combinations of cinnamic acids and anthranilate derivatives. Our data demonstrate the feasibility of rapidly producing a wide range of defined cinnamoyl anthranilates in yeast and underline a potential for the biological designed synthesis of naturally and non-naturally occurring molecules.


Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Biotecnología/métodos , Industria Farmacéutica/métodos , Saccharomyces cerevisiae/metabolismo , ortoaminobenzoatos/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Dianthus/enzimología , Dianthus/genética , Ingeniería Genética , Humanos , Redes y Vías Metabólicas/genética , Organismos Modificados Genéticamente , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética
15.
Mutat Res ; 722(2): 140-6, 2011 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-20633698

RESUMEN

Inactivation of DNA damage response mechanisms is associated with several disease syndromes, including cancer, aging and neurodegeneration. A major corrective pathway for alkylation or oxidative DNA damage is base excision repair (BER). As part of an effort to identify variation in DNA repair genes, we used the expressed sequence tag (EST) database to identify amino acid variation in Ape1, an essential gene in the BER repair pathway. Nucleotide substitutions were considered valid only if the amino acid changes were observed in at least two independent EST sequencing runs (i.e. two independent EST reports). In total eighty amino acid variants were identified for the Ape1 gene. Using software tools SIFT and PolyPhen, which predict impacts of amino acid substitutions on protein structure and function, twenty-six variants were predicted by both algorithms to be deleterious to protein function. Majority of these intolerant mutations such as V206C and F240S, lie within the core of the protein and may affect the stability and folding of Ape1, or in the case of N212H, N212K, and Y171N, are close to the enzyme's active site and could drastically affect its function. A few of the intolerant mutations, i.e., G178V and E217R, are surface residues and are far from the active site, and as such, the predicted effect on Ape1 stability or function is not evident. These variants are reagents for further protein function studies and molecular epidemiology studies of cancer susceptibility.


Asunto(s)
Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Polimorfismo de Nucleótido Simple , Algoritmos , Biología Computacional , Bases de Datos de Proteínas , Etiquetas de Secuencia Expresada , Humanos , Programas Informáticos
16.
Biochemistry ; 49(18): 3786-96, 2010 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-20377204

RESUMEN

Abasic (AP) sites are the most common lesions arising in genomic DNA. Repair of this potentially mutagenic DNA damage is initiated by the major apurinic/apyrimidinic endonuclease Ape1, which specifically recognizes and cleaves the DNA backbone 5' to the AP site. Ape1 is one of the major proteins in the base excision repair pathway (BER), and deletions in any of the BER proteins result in embryonic lethality. In this study, we employed fluorescence spectroscopy and in vitro mass spectrometric protein footprinting to investigate Ape1 conformational changes during various nucleoprotein interactions along its reaction pathway. Differences in intrinsic fluorescence emission spectra were observed during Ape1 protein's processing of the substrate, indicating possible conformational changes of the nucleoprotein complexes. To determine the protein domains that are involved in the putative conformational change, full-length Ape1 protein was probed with a lysine-reactive reagent (NHS-biotin) in the context of free protein and DNA-bound complexes. Protection patterns between pre- and postincision complexes revealed an increased susceptibility of lysine residues localized on the Ape1 surface that contacts the 3' end of the incised duplex (downstream of the incision site). We propose that the decreased protection results from Ape1 having a more relaxed grip on this section of the incised duplex to facilitate the handoff to the downstream BER enzyme. Protection of lysines (residues 24-35) in the N-terminal region was also observed in the intact AP-DNA-bound complex. These residues are part of the Ref1 domain which functions to regulate the activity of several transcription factors but to date has not been ascribed a DNA binding function. The reactivity of these Ref1 lysines was restored in the postincision complex. The differential protection patterns of lysines in the flexible N-terminal domain suggest a novel Ref1 conformational change concomitant with DNA binding and catalysis. It is likely that Ape1 employs this structural switch to mediate redox and nuclease activities. The ability of the Ape1-AP-DNA complex to recruit other BER proteins was also investigated by probing ternary complexes comprised of Ape1, DNA polymerase beta (Polbeta), and different BER DNA intermediates (abasic or gapped DNA). Our results suggest that Polbeta approaches the Ape1-DNA complex downstream of the incision site, displaces Ape1 DNA binding contacts (K227, K228, and K276), and in the process makes minimal interactions with lysine residues in the Ref1 domain.


Asunto(s)
Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN/genética , ADN/metabolismo , Daño del ADN , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Conformación Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína
17.
J Am Soc Mass Spectrom ; 17(3): 395-405, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16443364

RESUMEN

A systematic study of the dissociation patterns of crosslinked peptides analyzed by tandem mass spectrometry is reported. A series of 11-mer peptides was designed around either a polyalanine or polyglycine scaffold with arginine at the C terminus. One or two lysine residues were included at various locations within the peptides to effect inter- or intra-molecular crosslinking, respectively. Crosslinked species were generated with four commonly used amine-specific chemical crosslinking reagents: disuccinimidyl suberate (DSS), disuccinimidyl tartarate (DST), dithiobis(succinimidylpropionate) (DSP), and disuccinimidyl glutarate (DSG). The influence of precursor charge state, location of crosslink, and specific crosslinking reagent on the MS/MS dissociation pattern was examined. Observed trends in the dissociation patterns obtained for these species will allow for improvements to software used in the automated interpretation of crosslinked peptide MS/MS data.


Asunto(s)
Reactivos de Enlaces Cruzados/análisis , Reactivos de Enlaces Cruzados/química , Lisina/análisis , Lisina/química , Péptidos/análisis , Péptidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Sitios de Unión , Gases/análisis , Gases/química , Transición de Fase , Unión Proteica
19.
Science ; 306(5693): 79-86, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15459382

RESUMEN

Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for approximately 20% of global carbon fixation. We report the 34 million-base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand-base pair plastid and 44 thousand-base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.


Asunto(s)
Evolución Biológica , Diatomeas/genética , Ecosistema , Genoma , Análisis de Secuencia de ADN , Adaptación Fisiológica , Proteínas Algáceas/química , Proteínas Algáceas/genética , Proteínas Algáceas/fisiología , Animales , Núcleo Celular/genética , Cromosomas , ADN/genética , Diatomeas/química , Diatomeas/citología , Diatomeas/metabolismo , Metabolismo Energético , Hierro/metabolismo , Luz , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Mitocondrias/genética , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Fotosíntesis , Plastidios/genética , Mapeo Restrictivo , Alineación de Secuencia , Ácido Silícico/metabolismo , Simbiosis , Urea/metabolismo
20.
Chem Commun (Camb) ; (3): 322-3, 2003 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-12613593

RESUMEN

Biomolecules have been attached to porous silicon by a new linking method that forms a direct Si-C bond on the surface and retains the photoluminescence of the porous silicon.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Proteínas/química , Silicio , Biopolímeros/química , Cadaverina/química , Luminiscencia , Porosidad
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