A protein adaptor to locate a functional protein dimer on molecular switchboard.

The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard. We herein report a new adaptor to locate a protein dimer on the DNA origami scaffold based on a homodimeric basic-leucine zipper protein GCN4. Specific binding of GCN4 to programmed addresses on DNA origami and orthogonal targeting by GCN4- and zinc finger protein-based adaptors to the respective addresses on DNA origami were confirmed by gel electrophoretic and AFM analyses. Furthermore, a GCN4-fused homodimeric enzyme showed even higher activity than the wild type enzyme, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami.

Anti-prion activity of an RNA aptamer and its structural basis.

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP(Sc) level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP(C), resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.

Quantitative analysis of location- and sequence-dependent deamination by APOBEC3G using real-time NMR spectroscopy.

The human antiretroviral factor APOBEC3G (A3G) deaminates the newly synthesized minus strand of the human immunodeficiency virus 1 (HIV-1), which results in the abolition of the infectivity of virus-infectivity-factor (Vif)-deficient HIV-1 strains.1-6 A unique property of A3G is that it deaminates a CCC hot spot that is located close to the 5' end more effectively than one that is less close to the 5' end. However, the mechanism of this process is elusive as it includes nonspecific binding of A3G to DNA and sliding of A3G along the DNA strand. Therefore, this process cannot be analyzed by existing methods using the Michaelis-Menten theory. A new real-time NMR method has been developed to examine the nonspecific binding and the sliding processes explicitly, and it was applied to the analysis of the deamination by A3G. As a result, the location-dependent deamination can be explained by a difference in the catalytic rates that depend on the direction of the approach of A3G to the target cytidine. Real-time NMR experiments also showed that A3G deaminates CCCC tandem hotspots with little redundancy, which suggests that A3G efficiently mutates many CCC hotspots that are scattered throughout the HIV-1 genome.

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir.

Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially (15)N-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [(1)H, (15)N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) [14]. A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR=1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis.

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD(+) to NADP(+). In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.

Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes.

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO(2)-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5'-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa>Gua>adenine (Ade) approximately thymine (Thy)>cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.

Efficient bioethanol production by a recombinant flocculent Saccharomyces cerevisiae strain with a genome-integrated NADP+-dependent xylitol dehydrogenase gene.

The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.

Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives.

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.

Direct immobilization of DNA oligomers onto the amine-functionalized glass surface for DNA microarray fabrication through the activation-free reaction of oxanine.

Oxanine having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. By the chemical synthesis, a nucleotide unit of oxanine (Oxa-N) was incorporated into the 5'-end of probe DNA with or without the -(CH2)n- spacers (n = 3 and 12) and found to immobilize the probe DNA covalently onto the NH2-functionalized glass slide by one-pot reaction, producing the high efficiency of the target hybridization. The methylene spacer, particularly the longer one, generated higher efficiency of the target recognition although there was little effect on the amount of the immobilized DNA oligomers. The post-spotting treatment was also carried out under the mild conditions (at 25 or 42 degrees C) and the efficiencies of the immobilization and the target recognition were evaluated similarly, and analogous trends were obtained. It has also been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition. Immobilization of DNA oligomer by use of Oxa-N on the NH2-functionalized surface without any activation step would be employed as one of the advanced methods for generating DNA-conjugated solid surface.

Expression of protein engineered NADP+-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae.

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP(+)-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP(+)-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.

Bioethanol production from xylose by recombinant Saccharomyces cerevisiae expressing xylose reductase, NADP(+)-dependent xylitol dehydrogenase, and xylulokinase.

We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase.

Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.

Thermostabilization of Pichia stipitis xylitol dehydrogenase by mutation of structural zinc-binding loop.

Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type.

Chemical synthesis and thermodynamic characterization of oxanine-containing oligodeoxynucleotides.

Oxanine (Oxa, O), one of the major damaged bases from guanine generated by NO- or HNO2-induced nitrosative deamination, has been considered as a mutagen-potent lesion. For exploring more detailed properties of Oxa, large-scale preparation of Oxa-containing oligodeoxynucleotide (Oxa-ODN) with the desired base sequence is a prerequisite. In the present study, we have developed a chemical synthesis procedure of Oxa-ODNs and characterized thermodynamic properties of Oxa in DNA strands. First, 2'-deoxynucleoside of Oxa (dOxo) obtained from 2'-deoxyguanosine by HNO2-nitrosation was subjected to 5'-O-selective tritylation to give 5'-O-(4,4'-dimethoxytrityl)-dOxo (DMT-dOxo) with a maximum yield of 70%. Subsequently, DMT-dOxo was treated with conventional phosphoramidation, which resulted in DMT-dOxo-amidite monomer with a maximum yield of 72.5%. The amidite obtained was used for synthesizing Oxa-ODNs: the coupling yields for Oxa incorporation were over 93%. The prepared Oxa-ODNs were employed for analyzing the thermodynamic properties of DNA duplexes containing base-matches of O:N [N; C (cytosine), T (thymine), G (guanine) or A (adenine)]. Melting temperatures (Tm) and thermodynamic stability (DeltaG37(0)) were found to be lower by 6.83 approximately 13.41 degrees C and 2.643 approximately 6.047 kcal mol(-1), respectively, compared with those of oligodeoxynucleotides, which had the same base sequence except that O:N was replaced by G:C (wild type). It has also been found that Oxa-pairing with cytosine shows relatively high stability in DNA duplex compared with other base combinations. The orders of DeltaDeltaG37(0) were O:C > O:T > O:A > O:G. The chemical synthesis procedure and thermodynamic characteristics of Oxa-ODNs established here will be helpful for elucidating the biological significance of Oxa in relation to genotoxic and repair mechanisms.

Biophysical stability and enzymatic recognition of oxanine in DNA.

Oxanine (Oxa), which is one of the major products generated from guanine by nitrosative oxidation and is as long-lived as Gua in DNA, has been thought to be one of the major causes for NO-induced DNA damage. In the present study, using several synthetic Oxa-containing oligodeoxynucleotides, biophysical stability and enzymatic recognition of Oxa was investigated in DNA strands. It was found that Oxa did not mediate marked distortion in the whole DNA structure although Oxa pairing with 4 normal bases decreased thermal stability of the DNA duplexes compared to Gua:Cyt base pair. Regarding the responses of the DNA-relevant enzymes to Oxa, it was determined that Oxa was recognized as Gua except that DNA polymerases incorporated Thy as well as Cyt opposite Oxa. These results imply that Oxa tends to behave as a kind of naturally occurring base, Gua and therefore, would be involved in the genotoxic and cytotoxic threats of NO in cellular system.

A Novel Production Method for High-Fructose Glucose Syrup from Sucrose-Containing Biomass by a Newly Isolated Strain of Osmotolerant Meyerozyma guilliermondii.

One osmotolerant strain from among 44 yeast isolates was selected based on its growth abilities in media containing high concentrations of sucrose. This selected strain, named SKENNY, was identified as Meyerozyma guilliermondii by sequencing the internal transcribed spacer regions and partial D1/D2 large-subunit domains of the 26S ribosomal RNA. SK-ENNY was utilized to produce high-fructose glucose syrup (HFGS) from sucrose-containing biomass. Conversion rates to HFGS from 310-610 g/l of pure sucrose and from 75-310 g/l of sugar beet molasses were 73.5-94.1% and 76.2-91.1%, respectively. In the syrups produced, fructose yields were 89.4-100% and 96.5-100% and glucose yields were 57.6-82.5% and 55.3-79.5% of the theoretical values for pure sucrose and molasses sugars, respectively. This is the first report of employing M. guilliermondii for production of HFGS from sucrose-containing biomass.

Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA.

Improvement of a gene product by introducing mutations into the gene is usually applied for improving structural genes. In this study the procedure was applied for generation and improvement of a genetic signal to drive gene expression. By adding various concentrations of Mn2+ to the PCR reaction mixture, mutations were introduced into a DNA fragment at various ratios. An appropriate condition was employed to introduce mutations into a DNA fragment with no promoter activity. The mutated fragment was introduced at an upstream site of the lacZ gene in a plasmid vector to see if the fragment carries promoter activity. Lysate of an Escherichia coli transformant with the vector was assayed for beta-galactosidase expression as an indicator of the promoter activity. Mutated DNA fragments were generated by error prone PCR with a condition which leads to introduction of 1.5% of mutation into a DNA fragment during the process. The strongest promoter was chosen by beta-galactosidase assay after error prone PCR and subjected to another step of the PCR. These processes were repeated four times to improve its activity to 1.94-fold to that by the tac promoter. When the luciferase gene was expressed by the strongest promoters, a similar expression level was noted. These results indicate that by randomly introducing mutations into a DNA fragment, it is relatively easy to generate and improve a prokaryotic promoter.

A novel method for synthesis of silica nanoparticles.

A sequential method has been used, for the first time, to prepare monodisperse and uniform-size silica nanoparticles using ultrasonication by sol-gel process. The silica particles were obtained by hydrolysis of tetraethyl orthosilicate (TEOS) in ethanol medium and a detailed study was carried out on the effect of different reagents on particle sizes. Various-sized particles in the range 20-460 nm were synthesized. The reagents ammonia (2.8-28 mol L(-1)), ethanol (1-8 mol L(-1)), water (3-14 mol L(-1)), and TEOS (0.012-0.12 mol L(-1)) were used and particle size was examined under scanning electron microscopy and transmission electron microscopy. In addition to the above observations, the effect of temperature on particle size was studied. The results obtained in the present study are in agreement with the results observed for the electronic absorption behavior of silica particles, which was measured by UV-vis spectrophotometry.

Nitration of 2'-deoxyguanosine by a NO/O2 gas mixture: identification and characterization of N2-nitro-2'-deoxyguanosine.

[reaction: see text] A gas mixture of NO and O(2) was bubbled into 2'-deoxyguanosine solution at neutral pH and 37 degrees C. A novel nitrated nucleoside was generated in the reaction mixture in addition to 8-nitroguanine, 8-nitroxanthine, 2'-deoxyxanthosine, xanthine, and guanine. The novel nucleoside was identified as N(2)-nitro-2'-deoxyguanosine by spectrometric data.

Boost in bioethanol production using recombinant Saccharomyces cerevisiae with mutated strictly NADPH-dependent xylose reductase and NADP(+)-dependent xylitol dehydrogenase.

The xylose-fermenting recombinant Saccharomyces cerevisiae and its improvement have been studied extensively. The redox balance between xylose reductase (XR) and xylitol dehydrogenase (XDH) is thought to be an important factor in effective xylose fermentation. Using protein engineering, we previously successfully reduced xylitol accumulation and improved ethanol production by reversing the dependency of XDH from NAD(+) to NADP(+). We also constructed a set of novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis. In the present study, we constructed a set of recombinant S. cerevisiae carrying a novel set of mutated strictly NADPH-dependent XR and NADP(+)-dependent XDH genes with overexpression of endogenous xylulokinase (XK) to study the effects of complete NADPH/NADP(+) recycling on ethanol fermentation and xylitol accumulation. All mutated strains demonstrated reduced xylitol accumulation, ranging 34.4-54.7% compared with the control strain. Moreover, compared with the control strain, the two strains showed 20% and 10% improvement in ethanol production.