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At least 1,765 records · Page 98Linked to original sources
PubMed2006

New bioorganic reagents: evolved cyclohexanone monooxygenase--why is it more selective?

Four mutants of the cyclohexanone monooxygenase (CHMO) evolved as catalysts for Baeyer-Villiger oxidation of 4-hydroxycyclohexanone were investigated as catalysts for a variety of 4-substituted and 4,4-disubstituted cyclohexanones. Several excellent catalytic matches (mutant/substrate) were identified. The most important, however, is the finding that, in a number of cases, a mutant with a single exchange, Phe432Ser, was shown to be as robust and more selective as a catalyst than the wild-type CHMO. All biotransformations were performed on a laboratory scale, allowing full characterization of the products. The absolute configurations of two products were established. A model suggesting a possible role of the 432 serine residue in enantioselectivity control is proposed.

Amino Acid Substitution↗
PubMed1998

Kinetic preference for the 3'-5'-linked dimer in the reaction of guanosine 5'-phosphorylmorpholinamide with deoxyguanosine 5'-phosphoryl-2-methylimidazolide as a function of poly(C) concentration.

The formation of the internucleotide bond in diguanylate synthesis was studied in aqueous solution at pH 8 and 0.2 M Mg2+ in the presence and absence of polycytidylate, poly(C). The investigation was simplified by using guanosine 5'-phosphorylmorpholinamide, mor-pG, which can act only as a nucleophile, and deoxyguanosine 5'-phosphoryl-2-methylimidazolide, 2-MeImpdG, which can act only as an electrophile. The time-dependent product distribution was monitored by high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC/MS). In the absence of poly(C) the reaction between mor-pG and 2-MeImpdG yielded small amounts of the dimer mor-pGpdG with a regioselectivity of 2'-5':3'-5' = 3.5. In the presence of poly(C) dimer yields increased and a reversal in regioselectivity occurred; both effects were in proportion to the concentration of the polymer. The results can be quantitatively explained with the proposition that poly(C), acting as the template, catalyzes the reaction between template-bound monomers by about a factor of 4-5 over the reaction in solution and yields dimers with a regioselectivity of 2'-5':3'-5' approximately 0.33. These findings illustrate the intrinsic preference of guanosine monomers to correctly self-assemble on the appropriate template.

Chromatography, High Pressure Liquid↗
PubMed1999

Kinetics of template-directed pyrophosphate-linked dideoxyguanylate synthesis as a function of 2-MeImpdG and poly(C) concentration: insights into the mechanism.

Aqueous solutions of deoxyguanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpdG, yield primarily deoxyguanosine 5'-monophosphate, 5'dGMP, and pyrophosphate-linked dideoxyguanylate, dG5'ppdG, abbreviated G2p (see Chart 1). The initial rate of G2p formation, d[G2p]/dt in M h-1, determined at 23 degrees C, pH 7.8, 1.0 M NaCl and 0.2 M Mg2+ by timed high-performance liquid chromatography (HPLC) analysis, exhibits a second-order dependence on 2-MeImpdG concentration, [G]o, indicating a bimolecular mechanism of dimerization in the range 0.02 M < or = [G]o < or = 0.09 M. In the presence of polycytidylate, poly(C), G2p synthesis is accelerated and oligodeoxyguanylate products are formed by incorporation of 2-MeImpdG molecules. The kinetics of G2p formation as a function of both monomer and polymer concentration, expressed in C equivalents, were also determined under the above conditions and exhibited a complex behavior. Specifically, at a constant [poly(C)], values of d[G2p]/dt typically increased with [G]o with a parabolic upward curvature. At a constant [G]o, values of d[G2p]/dt increase with [poly(C)], but level off at the higher poly(C) concentrations. As [G]o increases this saturation occurs at a higher poly(C) concentration, a result opposite to expectation for a simple complexation of two reacting monomers with the catalyst prior to reaction. Nevertheless, these results are shown to be quantitatively consistent with a template-directed (TD) mechanism of dimerization where poly(C) acts as the template to bind 2-MeImpdG in a cooperative manner and lead, for the first time, to the formulation of principles that govern template-directed chemistry. Analysis of the kinetic data via a proposed TD cooperative model provides association constants for the affinity between polymer and monomer and the intrinsic reactivity of 2-MeImpdG toward pyrophosphate synthesis. To the best of our knowledge, poly(C)/2-MeImpdG is the first system that could serve as a textbook example of a TD reaction under conditions such that the template is fully saturated by monomers and under conditions that it is not.

Computer Simulation↗
PubMed2006

High-density, aligned SiO2 nanowire arrays: microscopic imaging of the unique growth style and their ultraviolet light emission properties.

High-density, free-standing SiO2 nanowire arrays were successfully fabricated by a simple chemical vapor deposition method through a controlled pattern of the micrometer-sized alloyed balls on the Si substrate combined with a local balanced and steady-state reaction vapor environment. The direct observation of temporal evolution of the SiO2 nanowire growth process via the microscopic imaging approach offers us amazing pictures related to the unique vapor-liquid-solid (VLS) growth styles. These novel results are beneficial to understanding the formation mechanism of silica nanowire arrays, and at the same time, they extend our knowledge of VLS growth phenomena. The stable and strong ultraviolet emission properties of the as-grown products are of significant interest for their potential applications related to nanoscale optoelectronic device including ultraviolet-light-emitting devices, etc.

Journal Article↗
PubMed2006

DNA-based molecular wires: multiple emission pathways of individual constructs.

The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.

DNA, Single-Stranded↗
PubMed2004

Nanoparticle-mediated epitaxial assembly of colloidal crystals on patterned substrates.

We have studied the assembly of 3-D colloidal crystals from binary mixtures of colloidal microspheres and highly charged nanoparticles on flat and epitaxially patterned substrates created by focused ion beam milling. The microspheres were settled onto these substrates from dilute binary mixtures. Laser scanning confocal microscopy was used to directly observe microsphere structural evolution during sedimentation, nanoparticle gelation, and subsequent drying. After microsphere settling, the nanoparticle solution surrounding the colloidal crystal was gelled in situ by introducing ammonia vapor, which increased the pH and enabled drying with minimal microsphere rearrangement. By infilling the dried colloidal crystals with an index-matched fluorescent dye solution, we generated full 3-D reconstructions of their structure including defects as a function of initial suspension composition and pitch of the patterned features. Through proper control over these important parameters, 3-D colloidal crystals were created with low defect densities suitable for use as templates for photonic crystals and photonic band gap materials.

Journal Article↗
PubMed1998

The case for a common ancestor: kinesin and myosin motor proteins and G proteins.

Recent studies have shown surprising structural and functional similarities between the motor domains of kinesin and myosin. Common features have also been described for motor proteins and G proteins. Despite these similarities, the evolutionary relationships between these proteins, even among the motor proteins, has not been obvious, since the topological connectivities of the core overlapping structural elements in these transducing proteins are not identical to one another. Using secondary structure topology, comparison of functional domains and active site chemistry as criteria for relatedness, we propose a set of rules for determining potential evolutionary relationships between proteins showing little or no sequence identity. These rules were used to explore the evolutionary relationship between kinesin and myosin, as well as between motor proteins and other phosphate-loop (P-loop) containing nucleotide-binding proteins. We demonstrate that kinesin and myosin show significant chemical conservations within and outside of the active site, and present an evolutionary scheme that produce their respective topologies from a hypothetical ancestral protein. We also show that, when compared with various other P-loop-containing proteins, the cytoskeletal motors are most similar to G proteins with respect to topology and active site chemistry. We conclude that kinesin and myosin, and possibly G proteins, are probably directly related via divergent evolution from a common core nucleotide-binding motif, and describe the likely topology of this ancestor. These proteins use similar chemical and physical mechanisms to both sense the state of the nucleotide bound in the active site, and then transmit these changes to protein partners. The different topologies can be accounted for by unique genetic insertions that add to the edge of a progenitor protein structure and do not disrupt the hydrophobic core.

Adenylate Kinase↗
PubMed1997

Dimerization in highly concentrated solutions of phosphoimidazolide activated mononucleotides.

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate. Here we report the self-condensation of nucleoside 5'-phosphate 2-methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2+ in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MeImpU and 2-MeImpC produce about 65% of oligomers including 4% of the 3',5'-linked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of internucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MeImpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.

Cytidine Monophosphate↗
PubMed1998

Studies on the lead-catalyzed synthesis of aldopentoses.

The object of this work was to find an efficient means of synthesizing ribose in a manner that could be considered prebiotic. The starting point for synthesis was an aqueous solution of formaldehyde. Heretofore the most frequently used catalyst for this purpose has been calcium hydroxide. Unfortunately this system produces a wide array of products in addition to ribose which constitutes 1% or less of the final product. Attempts were made to find more mild conditions under which the formaldehyde could be reacted. Magnesium hydroxide suspensions were used for this purpose. Formaldehyde does not yield any sugars when incubated in magnesium hydroxide suspensions alone. However, if the magnesium hydroxide suspension was supplemented with doubly charged lead salts and catalytic amounts of any intermediate in the prebiotic pentose pathway, aldopentoses accounted for 30 per cent or more of the final product. The presence of lead in the incubation mixture also accelerated a number of other reactions including the interconversion of the four common aldopentoses, ribose, arabinose, lyxose and xylose.

Acetophenones↗
PubMed2000

Bacterial cheaters.

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Directed Molecular Evolution↗
PubMed2000

Evolvability of an RNA virus is determined by its mutational neighbourhood.

The ubiquity of mechanisms that generate genetic variation has spurred arguments that evolvability, the ability to generate adaptive variation, has itself evolved in response to natural selection. The high mutation rate of RNA viruses is postulated to be an adaptation for evolvability, but the paradox is that whereas some RNA viruses evolve at high rates, others are highly stable. Here we show that evolvability in the RNA bacteriophage phi6 is also determined by the accessibility of advantageous genotypes within the mutational neighbourhood (the set of mutants one or a few mutational steps away). We found that two phi6 populations that were derived from a single ancestral phage repeatedly evolved at different rates and toward different fitness maxima. Fitness measurements of individual phages showed that the fitness distribution of mutants differed between the two populations. Whereas population A, which evolved toward a higher maximum, had a distribution that contained many advantageous mutants, population B, which evolved toward a lower maximum, had a distribution that contained only deleterious mutants. We interpret these distributions to measure the fitness effects of genotypes that are mutationally available to the two populations. Thus, the evolvability of phi6 is constrained by the distribution of its mutational neighbours, despite the fact that this phage has the characteristic high mutation rate of RNA viruses.

Bacteriophage phi 6↗
PubMed2001

Synthesizing life.

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Cell Compartmentation↗
PubMed2001

The bugs of war.

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Animals↗
PubMed2002

Genome shuffling leads to rapid phenotypic improvement in bacteria.

For millennia, selective breeding, on the basis of biparental mating, has led to the successful improvement of plants and animals to meet societal needs. At a molecular level, DNA shuffling mimics, yet accelerates, evolutionary processes, and allows the breeding and improvement of individual genes and subgenomic DNA fragments. We describe here whole-genome shuffling; a process that combines the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes normally associated with conventional breeding. We show that recursive genomic recombination within a population of bacteria can efficiently generate combinatorial libraries of new strains. When applied to a population of phenotypically selected bacteria, many of these new strains show marked improvements in the selected phenotype. We demonstrate the use of this approach through the rapid improvement of tylosin production from Streptomyces fradiae. This approach has the potential to facilitate cell and metabolic engineering and provide a non-recombinant alternative to the rapid production of improved organisms.

Directed Molecular Evolution↗