Evidence for an Enzyme-Catalyzed Rauhut-Currier Reaction during the Biosynthesis of Spinosyn A.

The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in Saccharopolyspora spinosa involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C-C bond formation. These results suggest that SpnL catalyzes a biological Rauhut-Currier reaction.

TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha.

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control.

Fha interaction with phosphothreonine of TssL activates type VI secretion in Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.

Effects of Chlorella sp. on nutrient treatment in cultures with different carbon to nitrogen ratios.

Chlorella sp. is often used in the treatment of wastewater to produce lipids, a practice which could go beyond wastewater treatment and be used to generate green energy. Our objectives here are to explore how the ratio of carbon to nitrogen (C/N) affects the removal of carbon and nitrogen in a wastewater treatment system, while simultaneously generating biomass and lipids. In this study, the C/N ratio is adjusted to 0.002, 6, 8, 10, 12 and 32. The results indicate that a C/N of 10 is sufficient to ensure the consumption of carbon and nitrogen, achieving the lowest concentration in the shortest culturing time (32 h). When nitrogen is lacking in the culture, there will be a slight decrease in the rate of carbon consumption which leads to a limitation of nitrogen and an increase in the lipid/cell density even at 96 h of culture time. The highest lipid content (0.57 g/L) and lipid increase rate (0.4 g/L) occurs with a C/N of 32. The greatest amount of biomass, 1.42 g/L is achieved when the C/N is 32. The carbon concentration is the main factor affecting the nitrogen consumption and the increase in the biomass and lipid content.

Structural delineation of MDC1-FHA domain binding with CHK2-pThr68.

Mammalian MDC1 interacts with CHK2 in the regulation of DNA damage-induced S-phase checkpoint and apoptosis, which is directed by the association of MDC1-FHA and CHK2-pThr68. However, different ligand specificities of MDC1-FHA have been reported, and no structure is available. Here we report the crystal structures of MDC1-FHA and its complex with a CHK2 peptide containing pThr68. Unlike other FHA domains, MDC1-FHA exists as an intrinsic dimer in solution and in crystals. Structural and binding analyses support pThr+3 ligand specificity and provide structural insight into MDC1-CHK2 interaction.

New ketamine analogue: 2-fluorodeschloro-N-ethyl-ketamine and its suggested metabolites.

Identifying new psychoactive substances (NPSs) and their metabolites is essential for regulating such substances for purposes of law enforcement and forensics. NPSs can be regulated on the basis of their chemical structures before they become a critical threat to society. Further, NPS metabolites can be targeted for analysis in urine, blood, and hair. This case study reports an incident in which 10 bags with approximately 15 g of crystalline material were seized from suspects by law enforcement officers and sent to the laboratory for confirmation. Gas chromatography-mass spectrometry, liquid chromatography-high-resolution mass spectrometry, and nuclear magnetic resonance (NMR) were employed to analyze these materials. The analyses revealed that the materials were a new ketamine analog, 2-fluorodeschloro-N-ethyl-ketamine (2-FDCNEK). Single-crystal X-ray diffraction (SXRD) analysis was also employed to confirm this result. In addition, metabolites of 2-FDCNEK were investigated using a fungal model and a urine sample from an abuser. The results suggest that 2-FDCNEK and 2-fluorodeschoro-norketamine are optimal metabolites for biological samples. This report presents the mass fragmentation, NMR analysis, and SXRD data of 2-FDCNEK. In addition, it provides suggestions regarding metabolites of 2-FDCNEK for law enforcement and forensic purposes, thereby facilitating the detection of this new ketamine analog.

Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily.

The rapid increase of multidrug resistance poses urgent threats to human health. Multidrug transporters prompt multidrug resistance by exporting different therapeutics across cell membranes, often by utilizing the H+ electrochemical gradient. MdfA from Escherichia coli is a prototypical H+ -dependent multidrug transporter belonging to the Major Facilitator Superfamily. Prior studies revealed unusual flexibility in the coupling between multidrug binding and deprotonation in MdfA, but the mechanistic basis for this flexibility was obscure. Here we report the X-ray structures of a MdfA mutant E26T/D34M/A150E, wherein the multidrug-binding and protonation sites were revamped, separately bound to three different substrates at resolutions up to 2.0 Å. To validate the functional relevance of these structures, we conducted mutational and biochemical studies. Our data elucidated intermediate states during antibiotic recognition and suggested structural changes that accompany the substrate-evoked deprotonation of E26T/D34M/A150E. These findings help to explain the mechanistic flexibility in drug/H+ coupling observed in MdfA and may inspire therapeutic development to preempt efflux-mediated antimicrobial resistance.

Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish.

In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO4-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time.

Diboron-Based Delayed Fluorescent Emitters with Orange-to-Red Emission and Superior Organic Light-Emitting Diode Efficiency.

For the application of organic light-emitting diodes (OLEDs) in lighting and panels, the basic requirement is to include a full spectrum color range. Compared with the development of blue and green luminophores in thermally activated delayed fluorescence (TADF) technology, the progress of orange-to-red materials is slow and needs further investigation. In this study, three diboron compound-based materials, dPhADBA, dmAcDBA, and SpAcDBA, were designed and synthesized by nucleophilic arylation of three amine donors on 9,10-diboraanthracene (DBA) in a two-step reaction. With increasing electron-donating ability, they show orange-to-red emission with TADF characteristics. The electroluminescence of these diboron compounds exhibits emissions λmax at 613, 583, and 567 nm for dPhADBA, dmAcDBA, and SpAcDBA, respectively. It is noteworthy that the rod-like D-A-D structures can achieve high horizontal ratios (84-86%) and outstanding device performance for orange-to-red TADF OLEDs: the highest external quantum efficiencies for dPhADBA, dmAcDBA, and SpAcDBA are 11.1 ± 0.5, 24.9 ± 0.5, and 30.0 ± 0.8%, respectively. Therefore, these diboron-based molecules offer a promising avenue for the design of orange-to-red TADF emitters and the development of highly efficient orange-to-red OLEDs.

Structure of an engineered multidrug transporter MdfA reveals the molecular basis for substrate recognition.

MdfA is a prototypical H+-coupled multidrug transporter that is characterized by extraordinarily broad substrate specificity. The involvement of specific H-bonds in MdfA-drug interactions and the simplicity of altering the substrate specificity of MdfA contradict the promiscuous nature of multidrug recognition, presenting a baffling conundrum. Here we show the X-ray structures of MdfA variant I239T/G354E in complexes with three electrically different ligands, determined at resolutions up to 2.2 Å. Our structures reveal that I239T/G354E interacts with these compounds differently from MdfA and that I239T/G354E possesses two discrete, non-overlapping substrate-binding sites. Our results shed new light on the molecular design of multidrug-binding and protonation sites and highlight the importance of often-neglected, long-range charge-charge interactions in multidrug recognition. Beyond helping to solve the ostensible conundrum of multidrug recognition, our findings suggest the mechanistic difference between substrate and inhibitor for any H+-dependent multidrug transporter, which may open new vistas on curtailing efflux-mediated multidrug resistance.

Evolution and biogeography of the endemic Roucela complex (Campanulaceae: Campanula) in the Eastern Mediterranean.

At the intersection of geological activity, climatic fluctuations, and human pressure, the Mediterranean Basin - a hotspot of biodiversity - provides an ideal setting for studying endemism, evolution, and biogeography. Here, we focus on the Roucela complex (Campanula subgenus Roucela), a group of 13 bellflower species found primarily in the eastern Mediterranean Basin. Plastid and low-copy nuclear markers were employed to reconstruct evolutionary relationships and estimate divergence times within the Roucela complex using both concatenation and species tree analyses. Niche modeling, ancestral range estimation, and diversification analyses were conducted to provide further insights into patterns of endemism and diversification through time. Diversification of the Roucela clade appears to have been primarily the result of vicariance driven by the breakup of an ancient landmass. We found geologic events such as the formation of the mid-Aegean trench and the Messinian Salinity Crisis to be historically important in the evolutionary history of this group. Contrary to numerous past studies, the onset of the Mediterranean climate has not promoted diversification in the Roucela complex and, in fact, may be negatively affecting these species. This study highlights the diversity and complexity of historical processes driving plant evolution in the Mediterranean Basin.

The bacteriohemerythrin from Methylococcus capsulatus (Bath): Crystal structures reveal that Leu114 regulates a water tunnel.

The bacteriohemerythrin (McHr) from Methylococcus capsulatus (Bath) is an oxygen carrier that serves as a transporter to deliver O2 from the cytosol of the bacterial cell body to the particulate methane monooxygenase residing in the intracytoplasmic membranes for methane oxidation. Here we report X-ray protein crystal structures of the recombinant wild type (WT) McHr and its L114A, L114Y and L114F mutants. The structure of the WT reveals a possible water tunnel in the McHr that might be linked to its faster autoxidation relative to hemerythrin in marine invertebrates. With Leu114 positioned at the end of this putative water tunnel, the hydrophobic side chain of this residue seems to play a prominent role in controlling the access of the water molecule required for autoxidation. This hypothesis is examined by comparing the autoxidation rates of the WT McHr with those of the L114A, L114Y and L114F mutants. The biochemical data are correlated with structural insights derived from the analysis of the putative water tunnels in the various McHr proteins provided by the X-ray structures.

Bacteriohemerythrin bolsters the activity of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath).

Recently, a native bacteriohemerythrin (McHr) has been identified in Methylococcus capsulatus (Bath). Both the particulate methane monooxygenase (pMMO) and McHr are over-expressed in cells of this bacterium when this strain of methanotroph is cultured and grown under high copper to biomass conditions. It has been suggested that the role of the McHr is to provide a shuttle to transport dioxygen from the cytoplasm of the cell to the intra-cytoplasmic membranes for consumption by the pMMO. Indeed, McHr enhances the activity of the pMMO when pMMO-enriched membranes are used to assay the enzyme activity. We find that McHr can dramatically improve the activity of pMMO toward the epoxidation of propylene to propylene oxide. The maximum activity is observed at a pMMO to McHr concentration ratio of 4:1, where we have obtained specific activities of 103.7nmol propylene oxide/min/mg protein and 122.8nmol propylene oxide/min/mg protein at 45°C when the turnover is driven by NADH and duroquinol, respectively. These results are consistent with the suggestion that the bacterium requires McHr to deliver dioxygen to the pMMO in the intra-cytoplasmic membranes to accomplish efficient catalysis of methane oxidation when the enzyme is over-expressed in the cells.