Bifunctional catalysts containing metal nanoparticles embedded on metal oxide supports are basic structures that exhibit high activity and efficiency for significant chemical reactions, such as hydrogenation, oxidation-reduction, and reforming. [1][2][3] In bifunctional structures, high-surface-area supports not only stabilize metal nanoparticles, but also generate new active components on a metal/metal-oxide interface, which facilitates chemical reactions.[4] Accordingly, the interactions between metal nanoparticles and supports should be precisely manipulated in order to maximize their synergetic effects. Metal particles should be dispersed evenly on the supports in order to prevent aggregation between particles in close proximity, and to maintain metal-support contact areas. Numerous processes have been developed for effective dispersion of metal nanoparticles in bifunctional catalysts. [5,6] However, severe particle aggregation during the reaction and surface inhomogeneity of the active particles still pose obstacles to the commercialization of catalysts in terms of high temperature stability and reusability. Recently, Somorjai et al. demonstrated a new concept of a ''nanoreactor'' system employing a Pt@CoO yolk/shell structure.[7] The Pt@CoO structure was generated from the reaction of Pt@Co core/shell particles with O 2 via the Kirkendall effect. The Pt cores in Pt@CoO could act as a catalyst for ethylene hydrogenation, whereas amorphous CoO shells transfer gas-phase reactants and products. [8] This nanoreactor system has many advantages in comparison to conventional catalysts embedded in bulk supports. First, each nanoparticle isolated by a solid shells has a relatively homogeneous environment around the particle surface. The outer shell structure also hinders the aggregation of neighboring particles, even under harsh reaction conditions. Second, the interaction of metal catalysts and supports is more effective than that of the bulk forms, feasibly leading to high catalytic activity. When common metal oxide supports (SiO 2 , TiO 2 , etc.) can substitute in the CoO shell structure, the resulting metal@metal oxide nanoreactor framework is expected to show intriguing catalytic behaviors for many gas and solution phase reactions.Similar yolk/shell-type structures have been known as ''nanorattles''. [9,10] Although such nanorattles are composed of metal particles and either silica or carbon shells, the synthetic procedures are multistep and complex, and achieving control over the metal core structures is challenging. Moreover, these nanorattles have yet to be employed as nanoreactor framework. In the present work, we fabricated a Au@SiO 2 yolk/shell structure through selective etching of metal cores from Au@SiO 2 core/shell particles (Scheme 1). The average size of the metal cores was readily tuned by using different amounts of etchants. This Au@SiO 2 framework was successfully employed in the catalytic reduction of p-nitrophenol as a model reaction. Gold nanoparticles were prepared by a modified polyol proces...

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Nanoreactor frameworks have many advantages over bulk catalyst structures in terms of providing a regular reaction environment and conformational stability. In this work, Au@SiO 2 nanoreactor frameworks were chemically modified to improve the catalytic efficiency of o-nitroaniline reduction. The porosity of silica shells was readily controlled by introducing C 18 TMS as a porogen with heat treatment. The diffusion rate of the silica layers was tuned from 5.9 × 10 -19 to 2.1 × 10 -18 m 2 s -1 , which directly altered the turnover frequency and rate constant of the reaction. Carboxylate functionality was introduced on the gold cores of Au@SiO 2 nanoreactors by 3-MPA addition. The reaction rate was enhanced by a maximum of 2.4 times compared to unfunctionalized catalysts through a strong interaction between carboxylate anions and o-nitroaniline. Totally, the rate constant of Au@SiO 2 yolk-shell nanoreactors exhibits a 13fold enhancement by diffusion and surface functionality control. These results indicate that the rational design of a nanoreactor framework with appropriate chemical functionalization can maximize the catalytic efficiency of various solution-phase reactions.

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The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolylhydroxylation in human hypoxia sensing has ancient origins.T he hypoxia-inducible transcription factor (HIF) is a major regulator of the response to limited oxygen availability in humans and other animals (1-3). A hypoxia-sensing component of the HIF system is provided by 2-oxoglutarate (2OG)-dependent and Fe(II)-dependent oxygenases, which catalyze prolyl-4-hydroxylation of HIF-α subunits, a posttranslational modification that enhances binding of HIF-α to the von Hippel-Lindau protein (pVHL), so targeting HIF-α for proteasomal degradation. The HIF prolyl-hydroxylases (PHDs) belong to a subfamily of 2OG oxygenases that catalyze prolyl-hydroxylation, which also includes the collagen prolyl-3-hydroxylases (CP3Hs) and prolyl-4-hydroxylases (CP4Hs) (4). Subsequently identified prolyl-hydroxylases include the ribosomal prolyl-hydroxylases (OGFOD1 and Tpa1), which catalyze ribosomal protein 23 prolyl-3-hydroxylation in many eukaryotes, and slime-mold enzymes, which catalyze prolyl-4-hydroxylation of Skp1, a ubiquitin ligase subunit (5-9). The HIF-PHD-VHL triad is likely present in all animals, but probably not in other organisms (3). However, structurally informed bioinformatic analyses imply the presence of PHD homologs in bacteria (10, 11), including in Pseudomonas spp, suggesting PHDs may have ancient origins. ResultsPseudomonas spp. Cont...

The site-specific incorporation of noncanonical monomers into polypeptides through genetic code reprogramming permits synthesis of bio-based products that extend beyond natural limits. To better enable such efforts, flexizymes (transfer RNA (tRNA) synthetase-like ribozymes that recognize synthetic leaving groups) have been used to expand the scope of chemical substrates for ribosome-directed polymerization. The development of design rules for flexizyme-catalyzed acylation should allow scalable and rational expansion of genetic code reprogramming. Here we report the systematic synthesis of 37 substrates based on 4 chemically diverse scaffolds (phenylalanine, benzoic acid, heteroaromatic, and aliphatic monomers) with different electronic and steric factors. Of these substrates, 32 were acylated onto tRNA and incorporated into peptides by in vitro translation. Based on the design rules derived from this expanded alphabet, we successfully predicted the acylation of 6 additional monomers that could uniquely be incorporated into peptides and direct N-terminal incorporation of an aldehyde group for orthogonal bioconjugation reactions.

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.semisynthesis | alpha-hemolysin | single-molecule chemistry | click chemistry

Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α- L -amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials.