Rapid Access to Divergent Fused Polycycles Via One-Pot A3 Coupling and Intramolecular Diels-Alder Reaction.

Divergent nitrogen-containing fused polycyclic ring systems are constructed from simple starting materials via a one-pot aldehyde-alkyne-amine (A3) coupling and intramolecular Diels-Alder reaction. This domino reaction directly furnishes linear 5/5/5 and 5/5/6, or nonlinear 5/5/6/5, polycyclic rings containing an oxa-bridged fused 5/5 bicycle and a 1,6-enyne substructure. One-step derivation of the oxa-bridged 5/5 bicycle leads to a polyfunctionalized 5/5 bicycle with tetrahydrofuran fused back-to-back to pyrroline or a 6/5 bicycle with the hexahydro-1H-isoindole structure, while cycloisomerizing the enyne substructure adds an extra fused 5-membered ring to afford functionalized linear 5/5/5/5 or 5/5/5/5/5 fused ring systems from selected substrates. In addition, the one-pot product can be designed so that the alkyne moiety is hydroalkoxylated to form an additional heterocyle in a linear 5/5/5/6 or nonlinear 5/5/6/5/5 ring system. This diversity-oriented synthetic approach thus allows rapid access to an under-explored structural space for discovery of new biological or non-biological activities or functions.

Discovery of P450-Modified Sesquiterpenoids Levinoids A-D through Global Genome Mining.

Cytochrome P450-modified bacterial terpenoids remain in a vast chemical space to be explored. In the present study, we conducted global genome mining of 223,829 bacterial genomes and identified 2892 bacterial terpenoid biosynthetic gene clusters (BGCs) with cytochrome P450 genes. Among these, we selected 562 with multiple P450 enzymes, which were further clustered as 355 gene cluster families by sequence similarity analysis. We then chose lev, a BGC from Streptomyces levis MCCC1A01616, for heterologous expression and discovered four new α-amorphene-type sesquiterpenoids, levinoids A-D (1-4). The structures and absolute configurations of these four new compounds were determined by employing extensive NMR analysis, NMR chemical shift calculations with DP4+, and ECD calculations. Furthermore, levinoid C (3) exhibited a moderate level of neuroprotective activity (EC50 = 21 µM) in the glutamate-induced excitotoxicity cell model. Our findings highlight the untapped chemical diversity of P450-modified bacterial terpenoids, opening new avenues for further exploration and discovery.

A vicinal oxygen chelate protein facilitates viral infection by triggering the unfolded protein response in Nicotiana benthamiana.

Vicinal oxygen chelate (VOC) proteins are members of an enzyme superfamily with dioxygenase or non-dioxygenase activities. However, the biological functions of VOC proteins in plants are poorly understood. Here, we show that a VOC in Nicotiana benthamiana (NbVOC1) facilitates viral infection. NbVOC1 was significantly induced by infection by beet necrotic yellow vein virus (BNYVV). Transient overexpression of NbVOC1 or its homolog from Beta vulgaris (BvVOC1) enhanced BNYVV infection in N. benthamiana, which required the nuclear localization of VOC1. Consistent with this result, overexpressing NbVOC1 facilitated BNYVV infection, whereas, knockdown and knockout of NbVOC1 inhibited BNYVV infection in transgenic N. benthamiana plants. NbVOC1 interacts with the basic leucine zipper transcription factors bZIP17/28, which enhances their self-interaction and DNA binding to the promoters of unfolded protein response (UPR)-related genes. We propose that bZIP17/28 directly binds to the NbVOC1 promoter and induces its transcription, forming a positive feedback loop to induce the UPR and facilitating BNYVV infection. Collectively, our results demonstrate that NbVOC1 positively regulates the UPR that enhances viral infection in plants.

A Photoactivatable Luminescent Motif through Ring-Flipping Isomerization for Multiple Photopatterning.

Photoactivatable luminescent materials have garnered enormous attention in the field of intelligent responsive materials, yet their design and applications remain challenging due to the limited variety of photoactivatable motifs. In the work described herein, we discovered a new photoactivatable luminescent motif that underwent ring-flipping isomerization under UV irradiation. The emission of this motif exhibited a rapid transformation from dark yellow to bright green, accompanied by a significant enhancement of quantum yield from 1.9% to 34.2%. Experimental and theoretical studies revealed that the effective intramolecular motion (EIM) was crucial to the distinct luminescence performance between two isomers. In addition, polymers containing this motif were achieved through a one-pot alkyne polymerization, exhibiting both photofluorochromic and photo-cross-linking properties. Furthermore, multiple types of photopatterning, including luminescent encryption, fluorescent grayscale imaging, and high-resolution photolithographic patterns, were realized. This work developed a new photoactivatable luminescent motif and demonstrated its potential applications in both small molecules and macromolecules, which will help in the future design of photoactivatable luminescent materials.

Identification and genomic characterization of a novel HIV-1 unique recombinant form (CRF01_AE/CRF07_BC) in Zhejiang Province, China.

Mutation and recombination are important mechanisms leading to the frequent evolution and genetic diversity of viruses as HIV-1. In this study, we identified the near full-length genomic characterization of a novel HIV-1 unique recombinant form (URF) strain (Sample ID: ZJ20202195/ZJ/CHN/2020, hereafter referred to as ZJ20202195) isolated during the HIV-1 molecular surveillance in 2020 in Zhejiang Province, China, through different recombination analysis tools and phylogenetic analysis. Our results amply proved that the near full-length genome (NFLG) sequence of ZJ20202195 was a novel HIV-1 unique recombinant form (URF) consisting of CRF01_AE and CRF07_BC subtype, and delimited three recombinant segments, of which the Segment I (HXB2:776-5559 nucleotide (nt)) and Segment III (HXB2:6224-9412 nt) were mainly originated from CRF01_AE cluster g4a strains prevalent in China and Segment II (HXB2:5560-6223 nt) was from CRF07_BC subtype. Overall, our findings provide insight and a scientific basis in the genetic diversity and accurate determination of HIV-1 recombinant strains in China.

Optimization of single-tube nested PCR for the detection of Echinococcus spp.

Echinococcosis is a serious zoonotic life-threatening parasitic disease caused by metacestodes of Echinococcus spp., and appropriate sensitive diagnosis and genotyping techniques are required to detect infections and study the genetic characterization of Echinococcus spp. isolates. In this study, a single-tube nested PCR (STNPCR) method was developed and evaluated for the detection of Echinococcus spp. DNA based on the COI gene. STNPCR was 100 times more sensitive than conventional PCR and showed the same sensitivity to common nested PCR (NPCR); but with a lower risk of cross-contamination. The limit of detection of the developed STNPCR method was estimated to be 10 copies/µL of the recombinant standard plasmids of Echinococcus spp. COI gene. In clinical application, 8 cyst tissue samples and 12 calcification tissue samples were analysed by conventional PCR with outer and inner primers and resulted in 100.00% (8/8) and 8.33% (1/12), 100.00% (8/8) and 16.67% (2/12) positive reactions, respectively, while STNPCR and NPCR were all able to identify the presence of genomic DNA in 100.00% (8/8) and 83.33% (10/12) of the same samples. Due to its high sensitivity combined with the potential for the elimination of cross-contamination, the STNPCR method was suitable for epidemiological investigations and characteristic genetic studies of Echinococcus spp. tissue samples. The STNPCR method can effectively amplify low concentrations of genomic DNA from calcification samples and cyst residues infected with Echinococcus spp. Subsequently, the sequences of positive PCR products were obtained, which were useful for haplotype analysis, genetic diversity, and evolution studies of Echinococcus spp., and understanding of Echinococcus spp. dissemination and transmission among the hosts.

Identification and prevalence of fluke infection in yak and Tibetan sheep around Qinghai Lake, China.

Liver flukes (Fasciola spp.) and rumen flukes (Paramphistomum spp.) are significant parasites in livestock worldwide, and Fasciola spp. are considered an important zoonotic parasite. To our knowledge, there are no reports on fluke species identification and epidemiological prevalence in yak and Tibetan sheep around Qinghai Lake, China. Therefore, this study aimed to identify the major fluke species and determine the prevalence of fluke infections among yak and Tibetan sheep in this area. A total of 307 fecal samples were collected and fluke eggs identified using morphology and molecular methods. Our study is the first to display that the predominant fluke species were F. hepatica and P. leydeni in yak and Tibetan sheep around Qinghai Lake. The overall prevalence of fluke infections in yak and Tibetan sheep was 57.7% (177/307). Specifically, the prevalences of F. hepatica and P. leydeni were 15.0% (46/307) and 31.6% (97/307), respectively, and the co-infection of both species was 11.1% (34/307). No significant difference existed in the prevalence of overall fluke infection between yak and Tibetan sheep (p < 0.05). However, F. hepatica prevalence was significantly different in yak and Tibetan sheep (p < 0.05) but not P. leydeni. The findings of this study provide useful information about the current status of natural fluke invasion in yak and Tibetan sheep around Qinghai Lake, which could be important for monitoring and controlling these parasites in the region.

Genome-wide phylogenetic and genetic evolutionary analyses of mitochondria in Hypoderma bovis and H. sinense on the Qinghai-Tibetan Plateau.

Hypoderma bovis (H. bovis) and Hypoderma sinense (H. sinense) are insects that cause hypodermosis in yaks and Bos taurus. Hypodermosis is a severe skin condition that not only impairs the development of local animal husbandry but also poses threats to human health as a zoonosis. The Qinghai-Tibetan Plateau (QTP) is known as the "Roof of the World." Its unique geographical environment and climate conditions have supported the growth of a wide range of mammals, providing favorable conditions for Hypoderma spp. to complete their life cycles. In this study, the whole mitochondrial genomes of H. bovis and H. sinense collected from the QTP were sequenced and phylogenetically analyzed. We found that the whole genomes of H. bovis and H. sinense are 16,283 bp and 16,300 bp in length, respectively. Both the H. bovis and H. sinense genomes have 37 mitochondrial genes, which include two rRNA genes (16S rRNA and 12S rRNA), 22 tRNA genes, the control region (D-loop region), the light chain replication initiation region, and 13 protein-coding genes (PCGs). The phylogenetic tree generated based on the 13 PCGs revealed close phylogenetic relationships between H. sinense, H. bovis, and Hypoderma lineatum. A similar result was also found in our phylogenetic analysis based on 18S rRNA and 28S rRNA. However, analysis of cytochrome oxidase subunit I (COI) showed cluster of H. bovis, H. sinense, and Cuterebra spp. on the same branch, all belonging to Oestridae. The differentiation time generated based on 13 PCGs indicates that H. bovis and H. sinense differentiated and formed ~4.69 million years ago (Mya) and ~4.06 Mya, respectively. This timing coincides with the differentiation and appearance of yak and Bos taurus in the Pliocene (~4.7 Mya), indicating that the parasites and mammals diverged in close temporal proximity. Of note, this period also witnessed a rapid uplift of the QTP, causing significant climate and environmental changes. Thus, we conjecture that the differentiation of Hypoderma spp. is potentially related to the differentiation of their host species, as well as climate changes caused by the uplift of the QTP. Overall, our study can provide valuable data to support further studies on the phylogeny and differentiation of Hypoderma spp. on the QTP.

Cry3Aa*SpyCatcher Fusion Crystals Produced in Bacteria as Scaffolds for Multienzyme Coimmobilization.

The production of Cry3Aa enzyme fusion crystals in Bacillus thuringiensis provides a direct method to immobilize individual enzymes and thereby improve their stability and recyclability. Nevertheless, many reactions require multiple enzymes to produce a desired product; thus a general strategy was developed to extend our Cry3Aa technology to multienzyme coimmobilization. Here, we report the direct production of particles comprising a modified Cry3Aa (Cry3Aa*) fused to SpyCatcher002 (Cry3Aa*SpyCat2) for coimmobilization of model enzymes MenF, MenD, and MenH associated with the biosynthesis of menaquinone. The resultant coimmobilized particles showed improved reaction rates compared to free enzymes presumably due to the higher local enzyme substrate concentrations and enhanced enzyme coupling made possible by colocalization. Furthermore, coimmobilization of these enzymes on Cry3Aa*SpyCat2 led to increased thermal stability and recyclability of the overall multienzyme system. These characteristics together with its overall simplicity of production highlight the benefits of Cry3Aa*SpyCat2 crystals as a platform for enzyme coimmobilization.

Listeria monocytogenes MenI Encodes a DHNA-CoA Thioesterase Necessary for Menaquinone Biosynthesis, Cytosolic Survival, and Virulence.

Listeria monocytogenes is a Gram-positive, intracellular pathogen that is highly adapted to invade and replicate in the cytosol of eukaryotic cells. Intermediate metabolites in the menaquinone biosynthesis pathway are essential for the cytosolic survival and virulence of L. monocytogenes, independent of the production of menaquinone (MK) and aerobic respiration. Determining which specific intermediate metabolite(s) are essential for cytosolic survival and virulence has been hindered by the lack of an identified 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) thioesterase essential for converting DHNA-CoA to DHNA in the MK synthesis pathway. Using the recently identified Escherichia coli DHNA-CoA thioesterase as a query, homology sequence analysis revealed a single homolog in L. monocytogenes, LMRG_02730 Genetic deletion of LMRG_02730 resulted in an ablated membrane potential, indicative of a nonfunctional electron transport chain (ETC) and an inability to aerobically respire. Biochemical kinetic analysis of LMRG_02730 revealed strong activity toward DHNA-CoA, similar to its E. coli homolog, further demonstrating that LMRG_02730 is a DHNA-CoA thioesterase. Functional analyses in vitro, ex vivo, and in vivo using mutants directly downstream and upstream of LMRG_02730 revealed that DHNA-CoA is sufficient to facilitate in vitro growth in minimal medium, intracellular replication, and plaque formation in fibroblasts. In contrast, protection against bacteriolysis in the cytosol of macrophages and tissue-specific virulence in vivo requires the production of 1,4-dihydroxy-2-naphthoate (DHNA). Taken together, these data implicate LMRG_02730 (renamed MenI) as a DHNA-CoA thioesterase and suggest that while DHNA, or an unknown downstream product of DHNA, protects the bacteria from killing in the macrophage cytosol, DHNA-CoA is necessary for intracellular bacterial replication.

Retinoblastoma cell-derived Twist protein promotes regulatory T cell development.

BACKGROUND: The development of tumor tissue-infiltrating regulatory T cell (Treg) is incompletely understood. This study investigates the role of retinoblastoma cell (Rbc)-derived Twist­related protein 1 (Twist) in the Treg development. METHODS: The surgically removed Rb tissues were collected. Rbcs were cultured with CD4+ T cells to assess the role of Rbc-derived Twist in the Treg generation. RESULTS: We found that more than 90% Rbcs expressed Twist. Foxp3+ Tregs were detected in the Rb tissues that were positively correlated with the Twist expression in Rbcs, negatively associated with Rb patient survival and sight survival. Treating Rbcs with hypoxia promoted the Twist expression that could be detected in the cytoplasm, nuclei and on the cell surface. Twist activated CD4+ T cells by binding the TLR4/myeloid differentiation factor 2 complex and promoted the transforming growth factor-ß-inducible early gene 1 product and Foxp3 expression. These Rbc-induced Foxp3+ Tregs showed immune-suppressive function on CD8+ T cell proliferation. CONCLUSIONS: Rbcs express Twist, that induces IL-4+ Foxp3+ Tregs; the latter can inhibit CD8+ cytotoxic T cell activities. Therefore, Twist may play an important role in the pathogenesis of Rb.

Performance of HIV detection in Zhejiang province in China: The Pareto principle at work.

BACKGROUND: Timely detection of HIV infection is critical for curbing the AIDS epidemic, and building an extensive and effective HIV laboratory network is of great importance. Therefore, improving quality management of the laboratory network and optimizing detection strategies are desirable research issues. METHODS: We assessed the applicability of the Pareto principle to HIV detection performance. We conducted a retrospective review of basic information and numbers of screening tests among an HIV laboratory network (1,452 laboratories) in Zhejiang province in 2014 and statistically analyzed HIV testing data for different population categories. RESULTS: Approximately, 80% of the cumulative HIV screening tests and positive screening tests originated from 17.3% (251/1,452) and 11.7% (170/1,452) of the laboratories in the whole province, respectively, and similar patterns were observed at the prefectural level. We found that the top five population screening categories (25%, 5/20) had the highest contribution (approximately 80%) to not only the number of screening tests (77.2%) but also the numbers of positive (76.4%) and confirmed positive tests (81.5%). CONCLUSIONS: The Pareto principle provides a method for identifying noteworthy laboratories to deliver prior quality supervision and developing highly efficient screening strategies that best suit local needs.

Deformable Metal-Organic Framework Nanosheets for Heterogeneous Catalytic Reactions.

The dynamic status near the surface of a catalyst can significantly affect the catalytic process, because the overall reaction rate depends on the mass velocity of product attachment and reactant detachment. As a dominant diffusion mechanism, molecular diffusion is known as a slow process that inhibits the fast contact between the reactants and the heterogeneous catalyst, which depresses catalytic conversion efficiency. Herein, we report a strategy that can break such a stagnant layer to facilitate the mass transport toward the catalyst surface, wherein Pd nanocubes (NCs) encapsulated in soft metal-organic framework (MOF) nanosheets are used as catalysts for the hydrogenation reactions. The soft MOF supports render deformable features to enhance mass transport across the Pd NCs, which is vital to enhance the catalyst performance. In combination with numerical simulations, we identify the deformable MOF driven by the shear force of flowing fluid to increase dye adsorption and catalytic conversion by 5- and 3-fold, respectively, as compared to a counterpart system containing nondeformable MOFs. The catalytic efficiency presents a volcano-type trend with the length-to-spacing ratio of MOF nanosheet being designed and reaches the maximum with a length-to-spacing ratio of 2:1. This technique provides unique opportunities to design a proof-of-concept self-propelled catalysis on the basis of a greater mechanistic understanding of heterogeneous catalytic reactions.

Substrate Recognition and Catalytic Mechanism of the Phosphate Acyltransferase PlsX from Bacillus subtilis.

Phosphate: acyl-acyl carrier protein (ACP) acyltransferase PlsX is a peripheral enzyme catalysing acyl transfer to orthophosphate in phospholipid synthesis. Little is known about how it recognises substrates and catalyses the acyl transfer. Here we show that its active site includes many residues lining a long, narrow gorge at the dimeric interface, two positive residues forming a positive ACP docking pad next to the interfacial gorge, and a number of strictly conserved residues significantly contributing to the catalytic activity. These findings suggest a substrate recognition mode and a catalytic mechanism that are different from those of phosphotransacetylases catalysing a similar acyl transfer reaction. The catalytic mechanism involves substrate activation and transition-state stabilization by two strictly conserved residues, Lys184 and Asn229. Another noticeable feature of the catalysis is the release of the acyl phosphate product near the membrane, which might facilitate its membrane insertion.

Structural characterization of Lanzhou lily (Lilium davidii var. unicolor) polysaccharides and determination of their associated antioxidant activity.

BACKGROUD: The Lanzhou lily (Lilium davidii var. unicolor) is the only Lilium species that is used for both culinary and medicinal purposes in China. Its bulbs contain various bioactive substances, such as polysaccharides, saponins and colchicine. Lanzhou lily polysaccharides are known to have anti-immunity, anti-tumor and anti-oxidation functions. RESULTS: The present study used a Box-Behnken design to optimize the ultrasound-assisted extraction of Lanzhou lily polysaccharides. Compared to other enzymes, trypsin significantly increased the polysaccharide yields, whereas the protein content of polysaccharides extracted with trypsin was the lowest. Monosaccharide mainly includes glucose (> 50%) and mannose (> 10%). 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity, chelating activity, total antioxidant capacity and hydroxyl radical scavenging activity of Lanzhou lily polysaccharides extracted with trypsin were stronger than those extracted without enzymes (control). Structural characteristics of Lanzhou lily polysaccharides extracted with trypsin and extracted without enzymes were characterized by scanning electron microscopy and nuclear magnetic resonance spectroscopy. When water extracted polysaccharide and trypsin extracted polysaccharide concentrations were 200 µg mL-1 , Raw264.7 proliferation rates were 101.69% and 159.41%, respectively. CONCLUSION: The Lanzhou lily polysaccharide was identified as α-(1 → 6)-d-glucan. Consequently, the effects of both potential antioxidant and proliferative activity of trypsin are significant. © 2020 Society of Chemical Industry.

Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing.

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

Two active site arginines are critical determinants of substrate binding and catalysis in MenD: a thiamine-dependent enzyme in menaquinone biosynthesis.

The bacterial enzyme MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral post-decarboxylation intermediates. The detailed mechanism of this intermediate chemistry, however, is still poorly understood, but of significant interest given that menaquinone is an essential electron transporter in many pathogenic bacteria. Here, we used site-directed mutagenesis, enzyme kinetic assays, and protein crystallography to reveal an active-inactive intermediate equilibrium in MenD catalysis and its modulation by two conserved active site arginine residues. We observed that these conserved residues play a key role in shifting the equilibrium to the active intermediate by orienting the C2-succinyl group of the intermediates through strong ionic hydrogen bonding. We found that when this interaction is moderately weakened by amino acid substitutions, the resulting proteins are catalytically competent with the C2-succinyl group taking either the active or the inactive orientation in the post-decarboxylation intermediate. When this hydrogen-bonding interaction was strongly weakened, the succinyl group was re-oriented by 180° relative to the native intermediate, resulting in the reversal of the stereochemistry at the reaction center that disabled catalysis. Interestingly, this inactive intermediate was formed with a distinct kinetic behavior, likely as a result of a non-native mode of enzyme-substrate interaction. The mechanistic insights gained from these findings improve our understanding of the new ThDP-dependent catalysis. More importantly, the non-native-binding site of the inactive MenD intermediate uncovered here provides a new target for the development of antibiotics.

Coenurosis of Yak, Bos grunniens, caused by Taenia multiceps: A Case Report with Molecular Identification in Qinghai Tibetan Plateau Area, China.

Coenurosis is an important zoonotic helminthic disease caused by the larval stage of the tapeworm Taenia multiceps. This parasite typically infects the brain of the intermediate hosts, including sheep, goat, cattle and even humans. We report a case of T. multiceps infection in a yak confirmed by clinical symptoms, morphological characteristics, and molecular and phylogenetic analyses. The coenurus was thin-walled, whitish, and spherical in shape with a diameter of 10 cm. The parasite species was identified as T. multiceps by PCR amplification and sequencing of the 18S rRNA, cox1 and nad1 genes. Three gene sequences all showed high homology (all above 97%) with the reference sequences from different hosts. Moreover, phylogenetic reconstructions with the 3 published Taenia gene sequences confirmed that the Qinghai yak isolate was closely related to T. multiceps. Although there are advanced diagnosis and treatment methods for coenurosis, early infection is difficult to diagnose. Importantly, the findings of yak infection case should not be ignored due to its zoonotic potential.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family.

EZH1 is an antipsychotic-sensitive epigenetic modulator of social and motivational behavior that is dysregulated in schizophrenia.

BACKGROUND: With the capacity to modulate gene networks in an environmentally-sensitive manner, the role of epigenetic systems in mental disorders has come under intense investigation. Dysregulation of epigenetic effectors, including microRNAs and histone-modifying enzymes, may better explain the role of environmental risk factors and the observed heritability rate that cannot be fully attributed to known genetic risk alleles. Here, we aimed to identify novel epigenetic targets of the schizophrenia-associated microRNA 132 (miR-132). METHODS: Histone modifications were quantified by immunodetection in response to viral-mediated overexpression of miR-132 while a luminescent reporter system was used to validate targets of miR-132 in vitro. Genome-wide profiling, quantitative PCR and NanoSting were used to quantify gene expression in post-mortem human brains, neuronal cultures and prefrontal cortex (PFC) of mice chronically exposed to antipsychotics. Following viral-mediated depletion of Enhancer of Zeste 1 (EZH1) in the murine PFC, behaviors including sociability and motivation were assessed using a 3-chambered apparatus and forced-swim test, respectively. RESULTS: Overexpression of miR-132 decreased global histone 3 lysine 27 tri-methylation (H3K27me3), a repressive epigenetic mark. Moreover, the polycomb-associated H3K27 methyltransferase, EZH1, is regulated by miR-132 and upregulated in the PFC of schizophrenics. Unlike its homolog EZH2, expression of EZH1 in the murine PFC decreased following chronic exposure to antipsychotics. Viral-mediated depletion of EZH1 in the mouse PFC attenuated sociability, enhanced motivational behaviors, and affected gene expression pathways related to neurotransmission and behavioral phenotypes. CONCLUSIONS: EZH1 is dysregulated in schizophrenia, sensitive to antipsychotic medications, and a brain-enriched miR-132 target that controls neurobehavioral phenotypes.