Exploring the origin of a unique mutant allele in twin-tail goldfish using CRISPR/Cas9 mutants.

Artificial selection has been widely applied to genetically fix rare phenotypic features in ornamental domesticated animals. For many of these animals, the mutated loci and alleles underlying rare phenotypes are known. However, few studies have explored whether these rare genetic mutations might have been fixed due to competition among related mutated alleles or if the fixation occurred due to contingent stochastic events. Here, we performed genetic crossing with twin-tail ornamental goldfish and CRISPR/Cas9-mutated goldfish to investigate why only a single mutated allele-chdS with a E127X stop codon (also called chdAE127X)-gives rise to the twin-tail phenotype in the modern domesticated goldfish population. Two closely related chdS mutants were generated with CRISPR/Cas9 and compared with the E127X allele in F2 and F3 generations. Both of the CRISPR/Cas9-generated alleles were equivalent to the E127X allele in terms of penetrance/expressivity of the twin-tail phenotype and viability of carriers. These findings indicate that multiple truncating mutations could have produced viable twin-tail goldfish. Therefore, the absence of polymorphic alleles for the twin-tail phenotype in modern goldfish likely stems from stochastic elimination or a lack of competing alleles in the common ancestor. Our study is the first experimental comparison of a singular domestication-derived allele with CRISPR/Cas9-generated alleles to understand how genetic fixation of a unique genotype and phenotype may have occurred. Thus, our work may provide a conceptual framework for future investigations of rare evolutionary events in domesticated animals.

Melatonin Enhanced Microglia M2 Polarization in Rat Model of Neuro-inflammation Via Regulating ER Stress/PPARδ/SIRT1 Signaling Axis.

Neuro-inflammation involves distinct alterations of microglial phenotypes, containing nocuous pro-inflammatory M1-phenotype and neuroprotective anti-inflammatory M-phenotype. Currently, there is no effective treatment for modulating such alterations. M1/M2 marker of primary microglia influenced by Melatonin were detected via qPCR. Functional activities were explored by western blotting, luciferase activity, EMSA, and ChIP assay. Structure interaction was assessed by molecular docking and LIGPLOT analysis. ER-stress detection was examined by ultrastructure TEM, calapin activity, and ERSE assay. The functional neurobehavioral evaluations were used for investigation of Melatonin on the neuroinflammation in vivo. Melatonin had targeted on Peroxisome Proliferator Activated Receptor Delta (PPARδ) activity, boosted LPS-stimulated alterations in polarization from the M1 to the M2 phenotype, and thereby inhibited NFκB-IKKß activation in primary microglia. The PPARδ agonist L-165,041 or over-expression of PPARδ plasmid (ov-PPARδ) showed similar results. Molecular docking screening, dynamic simulation approaches, and biological studies of Melatonin showed that the activated site was located at PPARδ (phospho-Thr256-PPARδ). Activated microglia had lowered PPARδ activity as well as the downstream SIRT1 formation via enhancing ER-stress. Melatonin, PPARδ agonist and ov-PPARδ all effectively reversed the above-mentioned effects. Melatonin blocked ER-stress by regulating calapin activity and expression in LPS-activated microglia. Additionally, Melatonin or L-165,041 ameliorated the neurobehavioral deficits in LPS-aggravated neuroinflammatory mice through blocking microglia activities, and also promoted phenotype changes to M2-predominant microglia. Melatonin suppressed neuro-inflammation in vitro and in vivo by tuning microglial activation through the ER-stress-dependent PPARδ/SIRT1 signaling cascade. This treatment strategy is an encouraging pharmacological approach for the remedy of neuro-inflammation associated disorders.

The multi-level regulation of clownfish metamorphosis by thyroid hormones.

Most marine organisms have a biphasic life cycle during which pelagic larvae transform into radically different juveniles. In vertebrates, the role of thyroid hormones (THs) in triggering this transition is well known, but how the morphological and physiological changes are integrated in a coherent way with the ecological transition remains poorly explored. To gain insight into this question, we performed an integrated analysis of metamorphosis of a marine teleost, the false clownfish (Amphiprion ocellaris). We show how THs coordinate a change in color vision as well as a major metabolic shift in energy production, highlighting how it orchestrates this transformation. By manipulating the activity of liver X regulator (LXR), a major regulator of metabolism, we also identify a tight link between metabolic changes and metamorphosis progression. Strikingly, we observed that these regulations are at play in the wild, explaining how hormones coordinate energy needs with available resources during the life cycle.

Targeting histone deacetylase-3 blocked epithelial-mesenchymal plasticity and metastatic dissemination in gastric cancer.

BACKGROUND AND PURPOSE: Histone deacetylase (HDAC) inhibitors (HDIs) can modulate the epithelial-mesenchymal transition (EMT) progression and inhibit the migration and invasion of cancer cells. Emerging as a novel class of anti-cancer drugs, HDIs are attracted much attention in the field of drug discovery. This study aimed to discern the underlying mechanisms of Honokiol in preventing the metastatic dissemination of gastric cancer cells by inhibiting HDAC3 activity/expression. EXPERIMENTAL APPROACH: Clinical pathological analysis was performed to determine the relationship between HDAC3 and tumor progression. The effects of Honokiol on pharmacological characterization, functional, transcriptional activities, organelle structure changes, and molecular signaling were analyzed using binding assays, differential scanning calorimetry, luciferase reporter assay, HDAC3 activity, ER stress response element activity, transmission electron microscopy, immune-blotting, and Wnt/ß-catenin activity assays. The in vivo effects of Honokiol on peritoneal dissemination were determined by a mouse model and detected by PET/CT tomography. KEY RESULTS: HDAC3 over-expression was correlated with poor prognosis. Honokiol significantly abolished HDAC3 activity (Y298) via inhibition of NFκBp65/CEBPß signaling, which could be reversed by the over-expression of plasmids of NFκBp65/CEBPß. Treatments with 4-phenylbutyric acid (a chemical chaperone) and calpain-2 gene silencing inhibited Honokiol-inhibited NFκBp65/CEBPß activation. Honokiol increased ER stress markers and inhibited EMT-associated epithelial markers, but decreased Wnt/ß-catenin activity. Suppression of HDAC3 by both Honokiol and HDAC3 gene silencing decreased cell migration and invasion in vitro and metastasis in vivo. CONCLUSIONS AND IMPLICATIONS: Honokiol acts by suppressing HDAC3-mediated EMT and metastatic signaling. By prohibiting HDAC3, metastatic dissemination of gastric cancer may be blocked. Conceptual model showing the working hypothesis on the interaction among Honokiol, HDAC3, and ER stress in the peritoneal dissemination of gastric cancer. Honokiol targeting HDAC3 by ER stress cascade and mitigating the peritoneal spread of gastric cancer. Honokiol-induced ER stress-activated calpain activity targeted HDAC3 and blocked Tyr298 phosphorylation, subsequently blocked cooperating with EMT transcription factors and cancer progression. The present study provides evidence to demonstrate that HDAC3 is a positive regulator of EMT and metastatic growth of gastric cancer cells. The findings here imply that overexpressed HDAC3 is a potential therapeutic target for honokiol to reverse EMT and prevent gastric cancer migration, invasion, and metastatic dissemination. • Honokiol significantly abolished HDAC3 activity on catalytic tyrosine 298 residue site. In addition, Honokiol-induced ER stress markedly inhibited HDAC3 expression via inhibition of NFκBp65/CEBPß signaling. • HDAC3, which is a positive regulator of metastatic gastric cancer cell growth, can be significantly inhibited by Honokiol. • Opportunities for HDAC3 inhibition may be a potential therapeutic target for preventing gastric cancer metastatic dissemination.

Aryl hydrocarbon receptor deficiency augments dysregulated microangiogenesis and diabetic retinopathy.

Diabetic retinopathy (DR) is a pathophysiologic vasculopathic process with obscure mechanisms and limited effective therapeutic strategies. Aryl hydrocarbon receptor (AhR) is an important regulator of xenobiotic metabolism and an environmental sensor. The aim of the present study was to investigate the role of AhR in the development of DR and elucidate the molecular mechanism of its downregulation. DR was evaluated in diabetes-induced retinal injury in wild type and AhR knockout (AhR-/-) mice. Retinal expression of AhR was determined in human donor and mice eyes by immunofluorescence since AhR activity was examined in diabetes. AhR knockout (AhRKO) mice were used to induce diabetes with streptozotocin, high-fat diet, or genetic double knockout with diabetes spontaneous mutation (Leprdb) (DKO; AhR-/-×Leprdb/db) for investigating structural, functional, and metabolic abnormalities in vascular and epithelial retina. Structural molecular docking simulation was used to survey the pharmacologic AhR agonists targeting phosphorylated AhR (Tyr245). Compared to diabetic control mice, diabetic AhRKO mice had aggravated alterations in retinal vasculature that amplified hallmark features of DR like vasopermeability, vascular leakage, inflammation, blood-retinal barrier breakdown, capillary degeneration, and neovascularization. AhR agonists effectively inhibited inflammasome formation and promoted AhR activity in human retinal microvascular endothelial cells and pigment epithelial cells. AhR activity and protein expression was downregulated, resulting in a decrease in DNA promoter binding site of pigment epithelium-derived factor (PEDF) by gene regulation in transcriptional cascade. This was reversed by AhR agonists. Our study identified a novel of DR model that target the protective AhR/PEDF axis can potentially maintain retinal vascular homeostasis, providing opportunities to delay the development of DR.

The real Nemo movie: Description of embryonic development in Amphiprion ocellaris from first division to hatching.

BACKGROUND: Amphiprion ocellaris is one of the rare reef fish species that can be reared in aquaria. It is increasingly used as a model species for Eco-Evo-Devo. Therefore, it is important to have an embryonic development table based on high quality images that will allow for standardized sampling by the scientific community. RESULTS: Here we provide high-resolution time-lapse videos to accompany a detailed description of embryonic development in A ocellaris. We describe a series of developmental stages and we define six broad periods of embryogenesis: zygote, cleavage, blastula, gastrula, segmentation, and organogenesis that we further subdivide into 32 stages. These periods highlight the changing spectrum of major developmental processes that occur during embryonic development. CONCLUSIONS: We provide an easy system for the determination of embryonic stages, enabling the development of A ocellaris as a coral reef fish model species. This work will facilitate evolutionary development studies, in particular studies of the relationship between climate change and developmental trajectories in the context of coral reefs. Thanks to its lifestyle, complex behavior, and ecology, A ocellaris will undoubtedly become a very attractive model in a wide range of biological fields.

Anemonefish, a model for Eco-Evo-Devo.

Anemonefish, are a group of about 30 species of damselfish (Pomacentridae) that have long aroused the interest of coral reef fish ecologists. Combining a series of original biological traits and practical features in their breeding that are described in this paper, anemonefish are now emerging as an experimental system of interest for developmental biology, ecology and evolutionary sciences. They are small sized and relatively easy to breed in specific husbandries, unlike the large-sized marine fish used for aquaculture. Because they live in highly structured social groups in sea anemones, anemonefish allow addressing a series of relevant scientific questions such as the social control of growth and sex change, the mechanisms controlling symbiosis, the establishment and variation of complex color patterns, and the regulation of aging. Combined with the use of behavioral experiments, that can be performed in the lab or directly in the wild, as well as functional genetics and genomics, anemonefish provide an attractive experimental system for Eco-Evo-Devo.

Embryonic and postembryonic development of the ornamental twin-tail goldfish.

BACKGROUND: Twin-tail ornamental goldfish have "bifurcated median fins," a peculiar morphology known to be caused by a mutation in the chdA gene. However, several ambiguities regarding the development of the phenotype remain due to a paucity of detailed observations covering the entire developmental timeframe. RESULTS: Here, we report a detailed comparative description of embryonic and postembryonic development for two representative twin-tail ornamental goldfish strains and single-tail common goldfish. Our observations reveal a polymorphic developmental process for bifurcated median fins; disrupted axial skeletal development at early larval stages; and modified bilateral location of the pelvic fin. CONCLUSIONS: Variations in development of bifurcated median fins and disrupted axial skeletal patterns reflect how artificial selection for adult morphological features influenced molecular developmental mechanisms during the domestication of twin-tail ornamental goldfish. The polymorphic appearance of bifurcated median fins also implies that, unlike previously proposed hypotheses, the development of these structures is controlled by molecular mechanisms independent of those acting on the pelvic fin. Our present findings will facilitate further study of how modifications of preexisting developmental systems may contribute to novel morphological features. Developmental Dynamics 248:251-283, 2019. © 2019 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Phyto-sesquiterpene lactone deoxyelephantopin and cisplatin synergistically suppress lung metastasis of B16 melanoma in mice with reduced nephrotoxicity.

BACKGROUND: Cisplatin (CP) is a chemotherapeutic drug for treating melanoma that also causes adverse side effects in cancer patients. PURPOSE: This study investigated the bioefficacy of a phytoagent deoxyelephantopin (DET) in inhibiting B16 melanoma cell activity, its synergism with CP against metastatic melanoma, and its capability to attenuate CP side effects in animals. METHODS: DET and CP bioactivities were assessed by MTT assay, isobologram analysis, time-lapse microscopy, migration and invasion assays, flow cytometry and western blotting. In vivo bioluminescence imaging was used to detect lung metastasis of B16 cells carrying COX-2 reporter gene in syngeneic mice. H&E staining and immunohistochemistry were used to evaluate the compound/drug efficacy and CP side effects. Nephrotoxicity caused by CP treatment in mice was evaluated by UPLC/ESI-QTOF MS - based metabolomics and haematometry. RESULT: DET, alone or in combination with cisplatin, inhibited B16 cell proliferation, migration, and invasion, and induced cell-cycle arrested at the G2/M phase and de-regulated cell-cycle mediators in cancer cells. In a murine B16COX-Luc metastatic allograft model, CP2 (2  mg/kg) treatment inhibited B16 lung metastasis accompanied by severe body weight loss, renal damage and inflammation, and haematological toxicity. DET10 and CP cotreatment (DET10 + CP1) or sequential treatment (CP2→DET10) significantly inhibited formation of pulmonary melanoma foci and reduced renal damage. DET pretreatment (Pre-DET10) or CP2→DET10 treatment had the longest survival (52  vs. 37 days for tumor control mice). CP treatment caused abnormally accumulated urea cycle metabolites and serotonin metabolite hippuric acid in renal tissues that were not seen with DET alone or in combination with CP. CONCLUSION: The CP and DET combination may be an effective intervention for melanoma with reduced side effects.

A novel allele of the goldfish chdB gene: Functional evaluation and evolutionary considerations.

The twin tail of ornamental goldfish is known to be caused by a nonsense mutation in one chordin paralogue gene. Our previous molecular studies in goldfish revealed that the ancestral chordin gene was duplicated, creating the chdA and chdB genes, and the subsequent introduction of a stop codon allele in the chdA gene ( chdA E127X ) caused the twin-tail morphology. The chdA E127X allele was positively selected by breeders, and the allele was genetically fixed in the ornamental twin-tail goldfish population. However, little is known about the evolutionary history of the chdB paralogue, begging the question: are there the functionally distinct alleles at the chdB locus, and if so, how did they evolve? To address these questions, we conducted molecular sequencing of the chdB gene from five different goldfish strains and discovered two alleles at the chdB gene locus; the two alleles are designated chdB 1 and chdB 2 . The chdB 1 allele is the major allele and was found in all investigated goldfish strains, whereas the chdB 2 allele is minor, having only been found in one twin-tail strain. Genetic analyses further suggested that these two alleles are functionally different with regard to survivability ( chdB 1 > chdB 2 ). These results led us to presume that in contrast to the chdA locus, the chdB locus has tended to be eliminated from the population. We also discuss how the chdB 2 allele was retained in the goldfish population, despite its disadvantageous function. This study provides empirical evidence of the long-term retention of a disadvantageous allele under domesticated conditions.

An alternative evolutionary pathway for the twin-tail goldfish via szl gene mutation.

The twin-tail of ornamental goldfish provides unique evolutionary evidence that the highly conserved midline localization of axial skeleton components can be changed by artificial selection. This morphological change is known to be caused by a nonsense mutation in one of the recently duplicated chordin genes, which are key players in dorsal-ventral (DV) patterning. Since all of the multiple twin-tail ornamental goldfish strains share the same mutation, it is reasonable to presume that this mutation occurred only once in domesticated goldfish. However, zebrafish with mutated szl gene (another DV patterning-related gene) also exhibit twin-tail morphology and higher viability than dino/chordin-mutant zebrafish. This observation raises the question of whether the szl gene mutation could also reproduce the twin-tail morphology in goldfish. Here we show that goldfish have at least two subfunctionalized szl genes, designated szlA and szlB, and depletion of these genes in single-fin goldfish was able to reproduce the bifurcated caudal fin found in twin-tail ornamental goldfish. Interestingly, several phenotypes were observed in szlA-depleted fish, while low expressivity of the twin-tail phenotype was observed in szlB-depleted goldfish. Thus, even though szl gene mutations may produce twin-tail goldfish, these szl gene mutations might not be favorable for selection in domestic breeding. These results highlight the uniqueness and rarity of mutations that are able to cause large-scale morphological changes, such as a bifurcated axial skeleton, with high viability and expressivity in natural and domesticated populations.

Open and closed evolutionary paths for drastic morphological changes, involving serial gene duplication, sub-functionalization, and selection.

Twin-tail goldfish strains are examples of drastic morphological alterations that emerged through domestication. Although this mutation is known to be caused by deficiency of one of two duplicated chordin genes, it is unknown why equivalent mutations have not been observed in other domesticated fish species. Here, we compared the chordin gene morphant phenotypes of single-tail goldfish and common carp (close relatives, both of which underwent chordin gene duplication and domestication). Morpholino-induced knockdown depleted chordin gene expression in both species; however, while knockdown reproduced twin-tail morphology in single-tail goldfish, it had no effect on common carp morphology. This difference can be explained by the observation that expression patterns of the duplicated chordin genes overlap completely in common carp, but are sub-functionalized in goldfish. Our finding implies that goldfish drastic morphological changes might be enhanced by the subsequent occurrence of three different types of evolutionary event (duplication, sub-functionalization, and selection) in a certain order.

The origin of the bifurcated axial skeletal system in the twin-tail goldfish.

Twin-tail goldfish possess a bifurcated caudal axial skeleton. The scarcity of this trait in nature suggests that a rare mutation, which drastically altered the mechanisms underlying axial skeleton formation, may have occurred during goldfish domestication. However, little is known about the molecular development of twin-tail goldfish. Here we show that the bifurcated caudal skeleton arises from a mutation in the chordin gene, which affects embryonic dorsal-ventral (DV) patterning. We demonstrate that formation of the bifurcated caudal axial skeleton requires a stop-codon mutation in one of two recently duplicated chordin genes; this mutation may have occurred within approximately 600 years of domestication. We also report that the ventral tissues of the twin-tail strain are enlarged, and form the embryonic bifurcated fin fold. However, unlike previously described chordin-deficient embryos, this is not accompanied by a reduction in anterior-dorsal neural tissues. These results provide insight into large-scale evolution arising from artificial selection.

Development of Cre-loxP technology in zebrafish to study the regulation of fish reproduction.

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

Characterization of tilapia (Oreochromis niloticus) viperin expression, and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle.

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.

Five different piscidins from Nile tilapia, Oreochromis niloticus: analysis of their expressions and biological functions.

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.

Taiwanin A inhibits MCF-7 cancer cell activity through induction of oxidative stress, upregulation of DNA damage checkpoint kinases, and activation of p53 and FasL/Fas signaling pathways.

This study investigates the anti-MCF-7 breast cancer cell effects and the underlying pharmacological activity and mechanism of taiwanin A, a major lignan isolated from Taiwania cryptomerioides. Our results show that taiwanin A time-dependently induced reactive oxygen species level and DNA damage in MCF-7 cells, which were likely activated kinases ataxia telangiectasia mutated (ATM) and checkpoint kinase (Chk). Taiwanin A could also up-regulate p53, phosphorylated p53, p21(Cip1), and p27(Kip1) and down-regulate the G(2)/M checkpoint cyclin-dependent kinase1 (Cdk1)-cyclin A/B, leading to induction of G(2)/M cell-cycle arrest in MCF-7 cells. Blockade of p53 gene expression by siRNA further demonstrated that the cell-cycle arrest induced by taiwanin A was p53-dependent. The FasL/Fas-mediated apoptotic signaling cascade was involved in taiwanin A-induced apoptosis via activation of caspases-10 and -7 (but not caspase-8), and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). In contrast, mitochondria-initiated apoptotic pathway was not involved. This is the first report to delineate novel mechanism of the action of taiwanin A against MCF-7 cells, suggesting this lignan may have value for development as an anti-breast cancer agent.

Structural and catalytic roles of residues located in beta13 strand and the following beta-turn loop in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase.

BACKGROUND: Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) is the only naturally occurring circularly permuted beta-glucanase among bacterial glucanases with reverse protein domains. We characterized the functional and structural significance of residues 200-209 located in the domain B of Fsbeta-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase. METHODS: Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study. RESULTS: Our kinetic data showed that D202N and D206N exhibited a 1.8- and 1.5-fold increase but G207N, G207-, F205L, N208G and T204F showed a 7.0- to 2.2-fold decrease, in catalytic efficiency (k(cat)/K(M)) compared to the wild-type enzyme. The comparative energy DeltaDeltaG(b) value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 degrees C over 10 min, whereas K200F was the most heat-sensitive enzyme. CONCLUSIONS: This study demonstrates that Phe205, Gly207, and Asn208 in the Type II turn of the connecting loop may play a role in the catalytic function of Fsbeta-glucanase. GENERAL SIGNIFICANCE: Residues 200-209 in Fsbeta-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.

Mutational and structural studies of the active-site residues in truncated Fibrobacter succinogenes1,3-1,4-beta-D-glucanase.

1,3-1,4-beta-D-Glucanases (EC 3.2.1.73) specifically hydrolyze beta-1,4-glycosidic bonds located prior to beta-1,3-glycosidic linkages in lichenan or beta-D-glucans. It has been suggested that truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TFsbeta-glucanase) can accommodate five glucose rings in its active site upon enzyme-substrate interaction. In this study, 12 mutant enzymes were created by mutating the conserved residues Gln70, Asn72, Gln81 and Glu85 proposed to bind to substrate subsites +1 and +2 and the catalytic properties of these mutants were determined. The most significant change in catalytic activity was observed on mutation of Gln70, with a 299-fold and 498-fold lower k(cat)/K(m) for the mutants Q70A and Q70I, respectively, compared with the wild-type enzyme. Mutagenesis, kinetic and structural studies revealed that the conserved residues surrounding the active site of TFsbeta-glucanase at substrate subsites +1 and +2 play an important role in its catalytic function, with the following order of importance: Gln70 > Asn72 > Glu85 > Gln81. The crystal structure of mutant E85I was determined at 2.2 A resolution. Further analysis of the E85I mutant structure revealed that the loop located at the concave site moved approximately 2 A from its position in the native enzyme complex without changing the core structure.

Crystal structure of truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase in complex with beta-1,3-1,4-cellotriose.

Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.