Transient cholinergic enhancement does not significantly affect either the magnitude or selectivity of perceptual learning of visual texture discrimination.

Perceptual learning (PL), often characterized by improvements in perceptual performance with training that are specific to the stimulus conditions used during training, exemplifies experience-dependent cortical plasticity. An improved understanding of how neuromodulatory systems shape PL promises to provide new insights into the mechanisms of plasticity, and by extension how PL can be generated and applied most efficiently. Previous studies have reported enhanced PL in human subjects following administration of drugs that increase signaling through acetylcholine (ACh) receptors, and physiological evidence indicates that ACh sharpens neuronal selectivity, suggesting that this neuromodulator supports PL and its stimulus specificity. Here we explored the effects of enhancing endogenous cholinergic signaling during PL of a visual texture discrimination task. We found that training on this task in the lower visual field yielded significant behavioral improvement at the trained location. However, a single dose of the cholinesterase inhibitor donepezil, administered before training, did not significantly impact either the magnitude or the location specificity of texture discrimination learning compared with placebo. We discuss potential explanations for discrepant findings in the literature regarding the role of ACh in visual PL, including possible differences in plasticity mechanisms in the dorsal and ventral cortical processing streams.

Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli.

P450-dependent biotransformations in Escherichia coli are attractive for the selective oxidation of organic molecules using mild and sustainable procedures. The overall efficiency of these processes, however, relies on how effectively the NAD(P)H cofactors derived from oxidation of the carbon source are utilized inside the cell to support the heterologous P450-catalyzed reaction. In this work, we investigate the use of metabolic and protein engineering to enhance the product-per-glucose yield (Y(PPG)) in whole-cell reactions involving a proficient NADPH-dependent P450 propane monooxygenase prepared by directed evolution [P450(PMO)R2; Fasan et al. (2007); Angew Chem Int Ed 46:8414-8418]. Our studies revealed that the metabolism of E. coli (W3110) is able to support only a modest propanol: glucose molar ratio (YPPG ~ 0.5) under aerobic, nongrowing conditions. By altering key processes involved in NAD(P)H metabolism of the host, considerable improvements of this ratio could be achieved. A metabolically engineered E. coli strain featuring partial inactivation of the endogenous respiratory chain (Δndh) combined with removal of two fermentation pathways (ΔadhE, Δldh) provided the highest Y(PPG) (1.71) among the strains investigated, enabling a 230% more efficient utilization of the energy source (glucose) in the propane biotransformation compared to the native E. coli strain. Using an engineered P450(PMO)R2 variant which can utilize NADPH and NADH with equal efficiency, we also established that dual cofactor specificity of the P450 enzyme can provide an appreciable improvement in Y(PPG). Kinetic analyses suggest, however, that much more favorable parameters (K(M), k(cat)) for the NADH-driven reaction are required to effectively compete with the host's endogenous NADH-utilizing enzymes. Overall, the metabolic/protein engineering strategies described here can be of general value for improving the performance of NAD(P)H-dependent whole-cell biotransformations in E. coli.

Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3.

Substrate engineered, achiral carboxylic acid derivative was biohydroxylated with various mutants of cytochrome P450 BM-3 to give two out of the four possible diastereoisomers in high de and ee. The BM-3 mutants exhibit up to 9200 total turnovers for hydroxylation of the engineered substrate, which without the protecting group is not transformed by this enzyme.

Synthesis and reactivity of a C3-symmetric trinuclear zinc(II) hydroxide catalyst efficient at phosphate diester transesterification.

Inspired by trinuclear Zn(II) sites in enzymatic systems, a ligand system containing three preorganized (2-pyridyl)methyl piperazine moieties anchored onto a rigid C3-symmetric triphenoxymethane platform has been developed for preorganizing three zinc ions into an environment conducive to intramolecular interaction. Zinc(II) binding by this ligand has been analyzed by means of potentiometric measurements in 50% (v/v) CH3CN-H2O solutions. Subsequently a C3-symmetric trinuclear Zn(II) hydroxide complex of the C3-symmetric ligand was synthesized and fully characterized using NMR spectroscopy and X-ray crystallography. This complex induces a 16,900-fold rate enhancement in the catalytic cyclization of the RNA model substrate, 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP, pH 6.7, 25 degrees C) over the uncatalyzed reaction with multiple catalyst turnovers. The observed differences in the pH-rate profile can be attributed to the varying concentration of various trinuclear zinc species. The trinuclear Zn(II) catalyst exhibits a higher hydrolytic activity compared to its mononuclear analogue. The reactivity and structural features of this trinuclear Zn(II) complex will be discussed.

CMPO-functionalized C3-symmetric tripodal ligands in liquid/liquid extractions: efficient, selective recognition of Pu(IV) with low affinity for 3+ metal ions.

Structural modifications of carbamoylmethylphosphine oxide (CPMO)-functionalized triphenoxymethane platforms are described, and the influence of these changes on the ability of the ligand to extract actinides from simulated acidic nuclear waste streams has been evaluated. The ligand system has been shown to have excellent binding efficiency and a selectivity for An(IV) in comparison to the a simple monomeric CMPO ligand under analogous conditions. Both the extraction efficiency and selectivity are strongly dependent on the flexibility and electronic properties of the ligating units in the triphenoxymethane construct. The Tb(III) and Bi(III) nitrate complexes of tris-CMPO derivatives have been isolated, and their structures were elucidated by NMR, ESI FT-ICR MS, and X-ray analysis, providing information on the interactions between metal ions and the tris-CMPO molecules.

Enantioselective epoxidation of terminal alkenes to (R)- and (S)-epoxides by engineered cytochromes P450 BM-3.

Cytochrome P450 BM-3 from Bacillus megaterium was engineered for enantioselective epoxidation of simple terminal alkenes. Screening saturation mutagenesis libraries, in which mutations were introduced in the active site of an engineered P450, followed by recombination of beneficial mutations generated two P450 BM-3 variants that convert a range of terminal alkenes to either (R)- or (S)-epoxide (up to 83 % ee) with high catalytic turnovers (up to 1370) and high epoxidation selectivities (up to 95 %). A biocatalytic system using E. coli lysates containing P450 variants as the epoxidation catalysts and in vitro NADPH regeneration by the alcohol dehydrogenase from Thermoanaerobium brockii generates each of the epoxide enantiomers, without additional cofactor.

Enhanced selectivity for actinides over lanthanides with CMPO ligands secured to a C(3)-symmetric triphenoxymethane platform.

A ligand system containing three preorganized carbamoylmethylphosphine oxide (CMPO) moieties anchored onto a rigid C(3)-symmetric triphenoxymethane platform has been developed for facile metal complexation and subsequent extraction from aqueous acidic nuclear waste streams. Intended to mimic the 3:1 CMPO-actinide stoichiometry of the extracted species in the TRUEX nuclear waste treatment process, the CMPO arms on this ligand are oriented such that all three CMPO moieties can cooperatively bind a metal ion. Extractions of simulated nuclear waste streams (10(-4) M metal in 1 M nitric acid) with solutions of this ligand in methylene chloride (10(-3) M) reveal a high affinity for the actinide thorium and a very low, but constant, affinity for the lanthanides across the series. Thorium and five lanthanide (lanthanum, cerium, neodymium, europium, and ytterbium) nitrate complexes of this ligand have been synthesized and fully characterized by X-ray crystallography, (1)H and (31)P NMR spectra, and FT-ICR-MS to elucidate the mechanism of this unique actinide selectivity. All six oxygen donors from the three CMPO arms of the ligand and one or two nitrate counterions coordinate these metals to afford 2+ cationic complexes in every case. Because of the large size of the ligand, both the thorium and lanthanide complexes present similarly charged and sized surfaces to the extraction solvents, but the thorium complex is extracted quantitatively over the lanthanide complexes. A possible rationale for this extraction behavior difference is presented and further illustrated by the extraction properties of this ligand system for the alkali metals (lithium, sodium, potassium, rubidium, and cesium) as picrate salts and by the solid- and solution-state structures of its lithium picrate complex.

Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3.

Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee). Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active (rates up to 400 min(-1)) and support thousands of product turnovers. The regio- and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.