Structural basis of homologous recombination.

Homologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase.

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase.

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.

A Thiamine-Dependent Enzyme Utilizes an Active Tetrahedral Intermediate in Vitamin K Biosynthesis.

Enamine is a well-known reactive intermediate mediating essential thiamine-dependent catalysis in central metabolic pathways. However, this intermediate is not found in the thiamine-dependent catalysis of the vitamin K biosynthetic enzyme MenD. Instead, an active tetrahedral post-decarboxylation intermediate is stably formed in the enzyme and was structurally determined at 1.34 Å resolution in crystal. This intermediate takes a unique conformation that allows only one proton between its tetrahedral reaction center and the exo-ring nitrogen atom of the aminopyrimidine moiety in the cofactor with a short distance of 3.0 Å. It is readily convertible to the final product of the enzymic reaction with a solvent-exchangeable proton at its reaction center. These results show that the thiamine-dependent enzyme utilizes a tetrahedral intermediate in a mechanism distinct from the enamine catalytic chemistry.

Molecular basis of the general base catalysis of an α/ß-hydrolase catalytic triad.

The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/ß-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/ß-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.

Microsecond protein folding events revealed by time-resolved fluorescence resonance energy transfer in a microfluidic mixer.

We demonstrate the combination of the time-resolved fluorescence resonance energy transfer (tr-FRET) measurement and the ultrarapid hydrodynamic focusing microfluidic mixer. The combined technique is capable of probing the intermolecular distance change with temporal resolution at microsecond level and structural resolution at Angstrom level, and the use of two-photon excitation enables a broader exploration of FRET with spectrum from near-ultraviolet to visible wavelength. As a proof of principle, we used the coupled microfluidic laminar flow and time-resolved two-photon excitation microscopy to investigate the early folding states of Cytochrome c (cyt c) by monitoring the distance between the tryptophan (Trp-59)-heme donor-acceptor (D-A) pair. The transformation of folding states of cyt c in the early 500 µs of refolding was revealed on the microsecond time scale. For the first time, we clearly resolved the early transient state of cyt c, which is populated within the dead time of the mixer (<10 µs) and has a characteristic Trp-59-heme distance of ∼31 Å. We believe this tool can find more applications in studying the early stages of biological processes with FRET as the probe.

All-in-one strategy to prepare molded biochar with magnetism from sewage sludge for high-efficiency removal of Cd(â ¡).

Biochar in powder could lead to the separation difficulties after using and easy dispersion by wind with non-necessary consumption during the practical application. The current method for preparing molded biochar is multi-step, tedious, and required exogenous reagents. Moreover, the dehydration of sewage sludge with high water content (>85%) causes expensive production cost, limiting its secondary utilization. Therefore, an "all-in-one" strategy was developed to prepare molded biochar with magnetism by using sewage sludge as endogenetic binder, water source, carbon source, as well as magnetic source, and biomass wastes as water moderator and pore-forming agent. The molded biochar showed high removal capacity towards Cd(Ⅱ) of 456.2 mg/g, which was 6 times higher than the commercial activated carbon in powder (69.1 mg/g). The excellent removal performance of the molded biochar was in linear correlation the O/C ratio (R2 =0.855), resulting in the complexation with Cd(Ⅱ). DFT calculations indicated the amounts and species of oxygen changed the electron distribution and electron-donation properties of biochar for Cd(Ⅱ). Moreover, the Na+ exchanges with Cd(Ⅱ) were also an important removal mechanism. This study provided a novel synthesis strategy for the molded biochar with both high particle density and high adsorption capability.

Novel Lipase Reactor based on Discontinuous Interfaces in Hydrogel-Organogel Hybrid Gel: A Preliminary Exploration.

According to the "interfacial activation" mechanism, constructing a sufficient interface is the key strategy for lipase-catalytic system designing. Based on the "infinite interface in finite three-dimensional space" logic, in the current study, poly(N,N-dimethylacrylamide) (PDMA)-polybutyl methacrylate (PBMA) hybrid gels were prepared by a two-step crosslinking strategy, subsequently constructed as lipase-interfacial catalytic systems. The results confirm that the PDMA-PBMA hybrid gels with "networks in pores" structures could swell both the aqueous phase and organic phase. The balance between water swelling and isooctane swelling, hybrid gel space (height control), and the lipase entry manner significantly affect the interface construction and consequently the catalytic efficiency. The enzyme-substrate contact rate affected by swelling leads to three catalytic stages. Considering the spatial barrier and distribution of lipases, a potential high-performance lipase reactor can be assembled from small-size, lamellar-like, and porous hybrid gels. The reactors also show good time storage and low temperature tolerance.

Active site binding and catalytic role of bicarbonate in 1,4-dihydroxy-2-naphthoyl coenzyme A synthases from vitamin K biosynthetic pathways.

1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes a carbon-carbon bond formation reaction in the biosynthesis of both vitamin K1 and K2. Bicarbonate is crucial to the activity of a large subset of its orthologues but lacks a clearly defined structural and mechanistic role. Here we determine the crystal structure of the holoenzymes from Escherichia coli at 2.30 Å and Synechocystis sp. PCC6803 at 2.04 Å, in which the bicarbonate cofactor is bound to the enzyme active site at a position equivalent to that of the side chain carboxylate of an aspartate residue conserved among bicarbonate-insensitive DHNA-CoA synthases. Binding of the planar anion involves both nonspecific electrostatic attraction and specific hydrogen bonding and hydrophobic interactions. In the absence of bicarbonate, the anion binding site is occupied by a chloride ion or nitrate, an inhibitor directly competing with bicarbonate. These results provide a solid structural basis for the bicarbonate dependence of the enzymatic activity of type I DHNA-CoA synthases. The unique location of the bicarbonate ion in relation to the expected position of the substrate α-proton in the enzyme's active site suggests a critical catalytic role for the anionic cofactor as a catalytic base in enolate formation.

Study on the stability and oral bioavailability of curcumin loaded (-)-epigallocatechin-3-gallate/poly(N-vinylpyrrolidone) nanoparticles based on hydrogen bonding-driven self-assembly.

The biological activity and absorption of curcumin (Cur) is limited in application due to its low water solubility, poorstabilityand rapid metabolism. In this work, Cur loaded (-)-epigallocatechin-3-gallate (EGCG)/poly(N-vinylpyrrolidone) (PVP) nanoparticles (CEP-NPs) was successfully fabricated via self-assembly driven by hydrogen bonding, providing with desirable Cur-loading efficiency, high stability, strong antioxidant capacity, and pH-triggered intestinal targeted release properties. Molecular dynamics simulations further indicated the Cur was coated with EGCG and PVP in CEP-NPs and high acid prolonged release property was attribute to low ionization degree of EGCG. Besides, the enhanced intestinal absorption of Cur was related to inhibition of Cur metabolism by EGCG, enhancement of cellular uptake and higher Caco-2 monolayer permeation. Pharmacokinetic study showed that the oral bioavailability presented nearly 12-fold increment. Therefore, this study provides a new horizon for improving the Cur utilization in food and pharmaceutical fields.

Stabilization of the second oxyanion intermediate by 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase of the menaquinone pathway: spectroscopic evidence of the involvement of a conserved aspartic acid.

1,4-Dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes an intramolecular Claisen condensation involving two oxyanion intermediates in the biosynthetic pathway of menaquinone, an essential respiration electron transporter in many microorganisms. Here we report the finding that the DHNA-CoA product and its analogues bind and inhibit the synthase from Escherichia coli with significant ultraviolet--visible spectral changes, which are similar to the changes induced by deprotonation of the free inhibitors in a basic solution. Dissection of the structure--affinity relationships of the inhibitors identifies the hydroxyl groups at positions 1 (C1-OH) and 4 (C4-OH) of DHNA-CoA or their equivalents as the dominant and minor sites, respectively, for the enzyme--ligand interaction that polarizes or deprotonates the bound ligands to cause the observed spectral changes. In the meantime, spectroscopic studies with active site mutants indicate that C4-OH of the enzyme-bound DHNA-CoA interacts with conserved polar residues Arg-91, Tyr-97, and Tyr-258 likely through a hydrogen bonding network that also includes Ser-161. In addition, site-directed mutation of the conserved Asp-163 to alanine causes a complete loss of the ligand binding ability of the protein, suggesting that the Asp-163 side chain is most likely hydrogen-bonded to C1-OH of DHNA-CoA to provide the dominant polarizing effect. Moreover, this mutation also completely eliminates the enzyme activity, strongly supporting the possibility that the Asp-163 side chain provides a strong stabilizing hydrogen bond to the tetrahedral oxyanion, which takes a position similar to that of C1-OH of the enzyme-bound DHNA-CoA and is the second high-energy intermediate in the intracellular Claisen condensation reaction. Interestingly, both Arg-91 and Tyr-97 are located in a disordered loop forming part of the active site of all available DHNA-CoA synthase structures. Their involvement in the interaction with the small molecule ligands suggests that the disordered loop is folded in interaction with the substrates or reaction intermediates, supporting an induced-fit catalytic mechanism for the enzyme.

Promotion of the anticancer activity of curcumin based on a metal-polyphenol networks delivery system.

Curcumin (Cur), a hydrophobic active pharmaceutical ingredient with high anticancer activity, has poor water solubility and low bioavailability. Although many delivery systems have been developed to improve their bioavailability, some limitation such as low drug loading efficiency and poor stability are still remained. The metal-polyphenol networks (MPNs) delivery system designed in this subject solved above problems and effectively improved the anticancer activity of Cur. The synthesized Cur@EGCG-Fe(III) is consisting of epigallocatechin gallate (EGCG), iron chloride (FeCl3) and Cur, and the well-designed structure endow Cur@EGCG-Fe(III) high loading efficiency, good water solubility and stability. After the Cur@EGCG-Fe(III) nanoparticles were internalized by MCF-7 cells, the Cur could be released in endo/lysosomal microenvironment (pH = 5.0), and the Cur delivery in the deep tumor could be realized. The distribution of Cur@EGCG-Fe(III) in MCF-7 cells was analyzed by laser confocal, and Cur@EGCG-Fe(III) could effectively deliver more Cur into MCF-7 cells in comparison with free Cur. In addition, the results of flow cytometry and western blot further indicated that Cur@EGCG-Fe(III) had a stronger ability to induce apoptosis than free Cur. Transwell cell migration and invasion experiments showed that Cur and EGCG-Fe(III) had a synergistic effect in inhibiting MCF-7 cell migration and invasion. In vitro hemolysis and in vivo experiments showed that the Cur@EGCG-Fe(III) had negligible effect on the blood environment and a great tumor-inhibition efficacy, indicating that the MPNs delivery system had a good blood compatibility and antitumor activity. Our results indicated that MPNs-coated Cur nanoparticle could be a new form of Cur delivery system for anticancer application.

Preferential hydrolysis of aberrant intermediates by the type II thioesterase in Escherichia coli nonribosomal enterobactin synthesis: substrate specificities and mutagenic studies on the active-site residues.

The type II thioesterase EntH is a hotdog fold protein required for optimal nonribosomal biosynthesis of enterobactin in Escherichia coli. Its proposed proofreading activity in the biosynthesis is confirmed by its efficient restoration of enterobactin synthesis blocked in vitro by analogs of the cognate precursor 2,3-dihydroxybenzoate. Steady-state kinetic studies show that EntH recognizes the phosphopantetheine group and the pattern of hydroxylation in the aryl moiety of its thioester substrates. Remarkably, it is able to distinguish aberrant intermediates from the normal one in the enterobactin assembly line by demonstrating at least 10-fold higher catalytic efficiency toward thioesters derived from aberrant aryl precursors without a para-hydroxyl group, such as salicylate. By structural comparison and site-directed mutagenesis, the thioesterase is found to possess an active site closely resembling that of the 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. strain SU and to involve an acidic residue (glutamate-63) as the catalytic base or nucleophile like all other hotdog thioesterases. In addition, the EntH specificities toward the substrate hydroxylation pattern are found to depend on the active-site histidine-54, threonine-64, serine-67, and methionine-68 with the selectivity significantly reduced or even reversed when they are individually replaced by alanine. These residues are likely responsible for differential interaction of the enzyme with the substrates which leads to distinction between the normal and aberrant precursors in the enterobactin assembly line. These results show that the type II thioesterase evolves its distinctive ability to recognize the aberrant intermediates from the versatile catalytic platform of hotdog proteins and suggests an active search mechanism for type II thioesterases in nonribosomal peptide synthesis.

A novel curcumin analog binds to and activates TFEB in vitro and in vivo independent of MTOR inhibition.

Autophagy dysfunction is a common feature in neurodegenerative disorders characterized by accumulation of toxic protein aggregates. Increasing evidence has demonstrated that activation of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal biogenesis, can ameliorate neurotoxicity and rescue neurodegeneration in animal models. Currently known TFEB activators are mainly inhibitors of MTOR (mechanistic target of rapamycin [serine/threonine kinase]), which, as a master regulator of cell growth and metabolism, is involved in a wide range of biological functions. Thus, the identification of TFEB modulators acting without inhibiting the MTOR pathway would be preferred and probably less deleterious to cells. In this study, a synthesized curcumin derivative termed C1 is identified as a novel MTOR-independent activator of TFEB. Compound C1 specifically binds to TFEB at the N terminus and promotes TFEB nuclear translocation without inhibiting MTOR activity. By activating TFEB, C1 enhances autophagy and lysosome biogenesis in vitro and in vivo. Collectively, compound C1 is an orally effective activator of TFEB and is a potential therapeutic agent for the treatment of neurodegenerative diseases.

Structural basis of the induced-fit mechanism of 1,4-dihydroxy-2-naphthoyl coenzyme A synthase from the crotonase fold superfamily.

1, 4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase fold enzyme with an implicated role of conformational changes in catalysis. We have identified these conformational changes by determining the structures of its Escherichia coli and Synechocystis sp. PCC6803 orthologues in complex with a product analog. The structural changes include the folding of an active-site loop into a ß-hairpin and significant reorientation of a helix at the carboxy terminus. Interestingly, a new interface is formed between the ordered loop and the reoriented helix, both of which also form additional interactions with the coenzyme A moiety of the ligand. Site-directed mutation of the amino acid residues involved in these ligand-induced interactions significantly diminishes the enzyme activity. These results suggest a catalytically essential induced-fit that is likely initiated by the enzyme-ligand interactions at the active site.