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The coral Acropora spp., known for its reef-building abilities, is a simultaneous hermaphroditic broadcast spawning species. Acropora spp. release gametes into seawater, activating sperm motility. This activation is mediated by adenylyl cyclase (AC) and protein kinase A (PKA). Notably, membrane-permeable cAMP (8-bromo-cAMP) promotes sperm motility activation of Acropora florida. While the signal transduction for PKA-dependent motility activation is highly conserved among animals, the downstream signaling of PKA remains unclear. In this study, we used mass spectrometry (MS) analyses to identify sperm proteins in the coral Acropora digitifera, as well as the serine/threonine residues of potential PKA substrates, and then, we investigated the conservation of these proteins from corals to vertebrates. We identified 148 sperm proteins of A. digitifera with typical PKA recognition motifs, namely RRXT and RRXS. We subsequently used ORTHOSCOPE to screen for orthologs encoding these 148 proteins from corals to vertebrates. Among the isolated orthologs, we identified positive selection in 48 protein-encoding genes from 18 Acropora spp. Subsequently, we compared the conservation rates of the PKA phosphorylation motif residues between the orthologs under positive and purifying selections. Notably, the serine residues of the orthologs under positive selection were more conserved. Therefore, adaptive evolution might have occurred in the orthologs of PKA substrate candidates from corals to vertebrates, accompanied by phosphorylation residue conservation. Collectively, our findings suggest that while PKA signal transduction, including substrates in sperm, may have been conserved, the substrates may have evolved to adapt to diverse fertilization conditions, such as synchronous broadcast spawning., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Coral terpenes are important molecules with numerous applications. Here, we describe a robust and simple method to produce coral terpene scaffolds at scale. As an example of the approach, here we discover, express, and characterize further klysimplexin R synthases, expanding the known enzymology of soft coral terpene cyclases. We hope that the underlying method described will enable widespread basic research into the functions of coral terpenes and their biosynthetic genes, as well as the commercial development of biomedically and technologically important molecules., (Copyright © 2024. Published by Elsevier Inc.)

Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses., (Copyright © 2024. Published by Elsevier Inc.)

It is widely known that metals can alter enzyme functioning, however, little is known about the mechanisms of metal toxicity in energy metabolism enzymes of corals. Thus, the present study had two objectives: firstly, we evaluated the activity of eight metabolic enzymes of the coral Mussismilia harttii to clarify metabolic functioning under field conditions. After that, we investigated the in vitro effect of copper (Cu) exposure in the activity of an enzyme representative of each metabolism stage. We evaluated enzymes involved in glycolysis (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK and lactate dehydrogenase, LDH), Krebs cycle (citrate synthase, CS and isocitrate dehydrogenase, IDH), electron transport chain (electron transport system activity, ETS) and pentose phosphate pathway (glucose-6-phosphate dehydrogenase, G6PDH). The in vitro tests were performed through contamination of the reaction medium using Cu concentrations of 0, 1.4, 3.7 and 14.2 μg L -1 . The results showed that M. harttii has elevated activity of HK, PK and CS in field conditions compared to the activity of other energy metabolism enzymes evaluated. Moreover, lower activities of LDH and ETS in exposed samples were observed. In conclusion, in field conditions this species has elevated aerobic metabolism and glucose may be an important energetic fuel. Also, exposure to Cu in vitro caused inhibition of LDH and ETS by direct binding., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata , a disease-sensitive reef-building coral, and Porites astreoides , a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a ( Orbicella faveolata caspase-3a) and PaCasp7a ( Porites astreoides caspase-7a), which we show to be D XX Dases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b ( Orbicella faveolata caspase-3b) and PaCasp3 ( Porites astreoides caspase-3), which we show to be V XX Dases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing D XX Dase is activated on death platforms but the protease does not directly activate the V XX Dase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Shrestha et al.)

Coral calcification is intricately linked to the chemical composition of the fluid in the extracellular calcifying medium (ECM), which is situated between the calcifying cells and the skeleton. Here we demonstrate that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) is expressed in calcifying cells of the coral Stylophora pistillata. Furthermore, pharmacological inhibition of sAC in coral microcolonies resulted in acidification of the ECM as estimated by the pH-sensitive ratiometric indicator SNARF, and decreased calcification rates, as estimated by calcein labeling of crystal growth. These results indicate that sAC activity modulates some of the molecular machinery involved in producing the coral skeleton, which could include ion-transporting proteins and vesicular transport. To our knowledge this is the first study to directly demonstrate biological regulation of the alkaline pH of the coral ECM and its correlation with calcification., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The combined effects of exposure to increasing temperature and copper (Cu) concentrations were evaluated in the zooxanthellate scleractinian coral Mussismilia harttii. Endpoints analyzed included activity of enzymes involved in glycolysis (pyruvate kinase, PK; lactate dehydrogenase, LDH), Krebs cycle (citrate synthase, CS; isocitrate dehydrogenase; IDH), electron transport chain (electron transport system, ETS) and pentose phosphate pathway (glucose-6-phosphate dehydrogenase, G6PDH). Coral polyps were kept under control conditions (25.0 ± 0.1 °C; 2.9 ± 0.7 μg/L Cu) or exposed to combined treatments of increasing temperature (26.6 ± 0.1 °C and 27.3 ± 0.1 °C) and concentrations of dissolved Cu (5.4 ± 0.9 and 8.6 ± 0.3 μg/L) for 4 and 12 days using a mesocosm system. PK activity was not affected by stressors. LDH, CS, IDH, ETS and G6PDH activities were temporally inhibited by stressors alone. CS, ETS and G6PDH activities remained inhibited by the combination of stressors after 12 days. Furthermore, all combinations between increasing temperature and exposure Cu were synergistic after prolonged exposure. Taken together, stressors applied alone led to temporary inhibitory effects on energy metabolism enzymes of the coral M. harttii, however, prolonged exposure reveals strong deleterious effects over the metabolism of corals due to the combination of stressors. The present study is the first one to give insights into the combined effects of increasing temperature and Cu exposure in the energy metabolism enzymes of a scleractinian coral. Findings suggest that moderate Cu contamination in future increasing temperature scenarios can be worrying for aerobic and oxidative metabolism of M. harttii., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A catalase-related allene oxide synthase (cAOS) or a hydroperoxide lyase (cHPL) fused together with an 8R-lipoxygenase is involved in the stress signaling of corals via an arachidonic acid pathway. cAOS gives rise to α-ketol and cyclopentenone, while cHPL catalyzes the cleavage of 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE) to C8-oxo acid and C12 aldehyde. In silico analysis of the substrate entry sites of highly identical coral cAOS and cHPL indicated that two positively charged residues of cAOS, K60 and K107, and the corresponding residues of cHPL, E60 and K107, may be involved in the anchoring of the carboxy group of polyunsaturated fatty acid (PUFA) hydroperoxides. A mutational analysis of cAOS and cHPL revealed that K60 or E60 and K107 were not necessary in the tethering of 8R-HpETE, however, the E60 of cHPL was essential in the productive binding of PUFA hydroperoxides. The substrate preferences of cAOS and cHPL were determined with hydroperoxy derivatives of C18, C20, C22 PUFAs, anandamide (AEA), 1-arachidonoyl glycerol (1-AG) and selected methylated substrates. Although cAOS and cHPL were able to metabolize different free PUFA substrates and arachidonoyl derivatives, only cHPL catalyzed the reaction with methylated PUFA hydroperoxides. The differences in the substrate binding and preferences between cAOS and cHPL can be explained by the distinct properties of their substrate entry sites. The current study demonstrated that homologous PUFA metabolizing enzymes may contribute to the versatile usage of the substrate pool., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The receptor guanylate cyclases (rGCs) in animals serve as sensitive chemoreceptors to detect both chemical and environmental cues. In reproduction, rGCs were shown to be expressed on sperm and serve as receptors for egg-derived sperm-activating and sperm-attracting factors in some echinoderms and mammals. However, sperm-associated rGCs have only been identified in some deuterostomes thus far, and it remains unclear how widely rGCs are utilized in metazoan reproduction. To address this issue, this study investigated the existence and expression of rGCs, particularly asking if rGCs are involved in the reproduction of a basal metazoan, phylum Cnidaria, using the stony coral Euphyllia ancora. Six paralogous rGCs were identified from a transcriptome database of E. ancora, and one of the rGCs, GC-A, was shown to be specifically expressed in the testis. Immunohistochemical analyses demonstrated that E. ancora GC-A protein was expressed in the spermatocytes and spermatids and eventually congregated on the sperm flagella during spermatogenesis. These findings suggest that GC-A may be involved in the regulation of sperm activity and/or functions (e.g., fertilization) in corals. This study is the first to perform molecular characterization of rGCs in cnidarians and provides evidence for the possible involvement of rGCs in the reproduction of basal metazoans.

Coral reefs are subjected to climate change and are severely impacted by human activities, with copper (Cu) being a relevant physiological stressor for corals at local scale. The ecological relevance of parameters measured at biochemical or cellular level is now considered an extremely important feature in environmental studies, and can be used as early warning signs of environmental degradation. In this context, the effects of acute exposure (96 h) to Cu were assessed on the maximum photochemical efficiency of zooxanthellae (Fv/Fm) and on the activity of key enzymes [carbonic anhydrase (CA) and Ca-ATPase] involved in coral physiology using the scleractinian coral Mussismilia harttii as a biological model. Corals were exposed to different concentrations of dissolved Cu (4.6-19.4 μg/L) using two different experimental approaches: a laboratory closed system and a marine mesocosm system. Fv/Fm values and Ca - ATPase activity were not affect by exposure to Cu in any of the exposure systems. However, a significant reduction in CA activity was observed in corals exposed to 11.9 and 19.4 μg Cu/L in the laboratory and at all concentrations of Cu tested in the mesocosm system (4.6, 6.0 and 8.5 μg/L). Based on the sensitivity of this enzyme to the short period of exposure to sublethal concentrations of Cu in both experimental approaches, the present study suggests the use of CA activity as a potential biomarker to be used in biomarker-based environmental monitoring programs in coral reefs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The asparaginyl hydroxylase, Factor Inhibiting HIF (FIH), is a cellular dioxygenase. Originally identified as oxygen sensor in the cellular response to hypoxia, where FIH acts as a repressor of the hypoxia inducible transcription factor alpha (HIF-α) proteins through asparaginyl hydroxylation, FIH also hydroxylates many proteins that contain ankyrin repeat domains (ARDs). Given FIH's promiscuity and the unclear functional effects of ARD hydroxylation, the biological relevance of HIF-α and ARD hydroxylation remains uncertain. Here, we have employed evolutionary and enzymatic analyses of FIH, and both HIF-α and ARD-containing substrates, in a broad range of metazoa to better understand their conservation and functional importance. Utilising Tribolium castaneum and Acropora millepora, we provide evidence that FIH from both species are able to hydroxylate HIF-α proteins, supporting conservation of this function beyond vertebrates. We further demonstrate that T. castaneum and A. millepora FIH homologs can also hydroxylate specific ARD proteins. Significantly, FIH is also conserved in several species with inefficiently-targeted or absent HIF, supporting the hypothesis of important HIF-independent functions for FIH. Overall, these data show that while oxygen-dependent HIF-α hydroxylation by FIH is highly conserved in many species, HIF-independent roles for FIH have evolved in others., Competing Interests: The authors have declared that no competing interests exist.

Microplastics are widespread emerging contaminants that have been found globally in the marine and freshwater ecosystem, but there is limited knowledge regarding its impact on coral reef ecosystem and underpinning mechanism. In the present study, using Pocillopora damicornis as a model, we investigated cytological, physiological, and molecular responses of a scleractinian coral to acute microplastic exposure. No significant changes were observed in the density of symbiotic zooxanthellae during the entire period of microplastic exposure, while its chlorophyll content increased significantly at 12 h of microplastic exposure. We observed significant increases in the activities of antioxidant enzymes such as superoxide dismutase and catalase, significant decrease in the detoxifying enzyme glutathione S-transferase and the immune enzyme alkaline phosphatase, but no change in the other immune enzyme phenoloxidase during the whole experiment period. Transcriptomic analysis revealed 134 significantly up-regulated coral genes at 12 h after the exposure, enriched in 11 GO terms mostly related to stress response, zymogen granule, and JNK signal pathway. Meanwhile, 215 coral genes were significantly down-regulated at 12 h after exposure, enriched in 25 GO terms involved in sterol transport and EGF-ERK1/2 signal pathway. In contrast, only 12 zooxanthella genes exhibited significant up-regulation and 95 genes down-regulation at 12 h after the microplastic exposure; genes regulating synthesis and export of glucose and amino acids were not impacted. These results suggest that acute exposure of microplastics can activate the stress response of the scleractinian coral P. damicornis, and repress its detoxification and immune system through the JNK and ERK signal pathways. These demonstrate that microplastic exposure can compromise the anti-stress capacity and immune system of the scleractinian coral P. damicornis, despite the minimal impact on the abundance and major photosynthate translocation transporters of the symbiont in the short term., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Atmospheric concentration of carbon dioxide (CO 2 ) is increasing at an unprecedented rate and subsequently leading to ocean acidification. Concomitantly, ocean warming is intensifying, leading to serious and predictable biological impairments over marine biota. Reef-building corals have proven to be very vulnerable to climate change, but little is known about the resilience of non-reef-building species. In this study, we investigated the effects of ocean warming and acidification on the antioxidant enzyme activity (CAT-catalase, and GST-glutathione S-transferase), lipid peroxidation (using malondialdehyde, MDA-levels as a biomarker) and heat shock response (HSP70/HSC70 content) of the octocoral Veretillum cynomorium. After 60 days of acclimation, no mortalities were registered in all treatments. Moreover, CAT and GST activities, as well as MDA levels, did not change significantly under warming and/or acidification. Heat shock response was significantly enhanced under warming, but high CO 2 did not have a significant effect. Contrasting to many of their tropical coral-reef relatives, our findings suggest that temperate shallow-living octocorals may be able to physiologically withstand future conditions of increased temperature and acidification.

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes used by living organisms to accelerate the CO₂ hydration/dehydration reaction at rates dramatically high compared to the uncatalyzed reaction. These enzymes have different isoforms and homologues and can be found in the form of cytoplasmic, secreted or membrane-bound proteins. CAs play a role in numerous physiological processes including biomineralization and symbiosis, as is the case in reef-building corals. Previously, molecular and biochemical data have been obtained at the molecular level in the branching coral Stylophora pistillata for two coral isoforms which differ significantly in their catalytic activity and susceptibility to inhibition with anions and sulfonamides. More recently it has been determined that the genome of S. pistillata encodes for 16 CAs. Here, we cloned, expressed, purified and characterized a novel α-CA, named SpiCA3, which is cytoplasmic and ubiquitously expressed in all the cell layers including the calcifying cells. SpiCA3 is the most effective CA among the coral isoforms investigated and the most efficient catalyst known up to date in Metazoa. We also investigated the inhibition profiles of SpiCA3 and compared it with those obtained for the two other isoforms in the presence of inorganic anions and other small molecules known to interfere with metalloenzymes. These results suggest that S. pistillata has adapted its CA isoforms to achieve the physiological functions in different physicochemical microenvironments.

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis , looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

CruCA4 is a secreted isoform of the α-carbonic anhydrase (CA, EC 4.2.1.1) family, which has been identified in the octocoral Corallium rubrum. This enzyme is involved in the calcification process leading to the formation of the coral calcium carbonate skeleton. We report here experiments performed on the recombinant CruCA4 with the technique of protonography that can be used to detect in a simple way the enzyme activity. We have also investigated the inhibition profile of CruCA4 with one major class of CA inhibitors, the inorganic anions. A range of weak and moderate inhibitors have been identified having K I in the range of 1-100 mM, among which the halides, pseudohalides, bicarbonate, sulfate, nitrate, nitrite, and many complex inorganic anions. Stronger inhibitors were sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethylditiocarbamate, which showed a better affinity for this enzyme, with K I in the range of 75 μM-0.60 mM. All these anions/small molecules probably coordinate to the Zn(II) ion within the CA active site as enzyme inhibition mechanism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The cDNA sequence of arginine kinase (AK) from the precious coral Corallium rubrum was assembled from transcriptome sequence data, and the deduced amino acid sequence of 364 residues was shown to conserve the structural features characteristic of AK. Based on the amino acid sequence, the DNA coding C. rubrum AK was synthesized by overlap extension PCR to prepare the recombinant enzyme. The following kinetic parameters were determined for the C. rubrum enzyme: K a Arg (0.10 mM), K ia Arg (0.79 mM), K a ATP (0.23 mM), K ia ATP (2.16 mM), and k cat (74.3 s -1 ). These are comparable with the kinetic parameters of other AKs. However, phylogenetic analysis suggested that the C. rubrum AK sequence has a distinct origin from that of other known cnidarian AKs with unusual two-domain structure. Using oligomers designed from the sequence of C. rubrum AK, the coding region of genomic DNA of another coral Paracorallium japonicum AK was successfully amplified. Although the nucleotide sequences differed between the two AKs at 14 positions in the coding region, all involved synonymous substitutions, giving the identical amino acid sequence. The P. japonicum AK gene contained one intron at a unique position compared with other cnidarian AK genes. Together with the observations from phylogenetic analysis, the comparison of exon/intron organization supports the idea that two distinct AK gene lineages are present in cnidarians. The difference in the nucleotide sequence between the coding regions of C. rubrum and P. japonicum AKs was 1.28%, which is twice that (0.54%) of mitochondrial DNA, is consistent with the general observation that the mitochondrial genome evolves slower than the nuclear one in cnidarians.

Lipoxygenases (LOXs), participating in inflammatory processes and cancer, are a family of enzymes with high potential as drug targets. Various allosteric effects have been observed with different LOX isozymes (e.g. lipid/ATP binding, phosphorylation), yet there is a lot of uncertainty concerning the regulation of these enzymes. It has been recently found that a number of LOXs form dimers, extending the list of possible allosteric mechanisms with oligomerization. Coral 11R-LOX is, unlike several mammalian counterparts, a stable dimer in solution facilitating quaternary structure studies that demand high sample homogeneity. By combining previous crystallographic data of 11R-LOX with small-angle X-ray scattering and chemical cross-linking, we were able to narrow down the possible dimerization interfaces, and subsequently determined the correct assembly by site-directed mutagenesis of potential contacting residues. The region of interest is located in the vicinity of an α+β formation in the catalytic domain, also coined the PDZ-like domain. Being situated just between the active site and the dimer interface, our results further implicate this putative subdomain in the regulation of LOXs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, we cloned and characterized eleven serine and aspartate racemases (SerR and AspR, respectively) from animals. These SerRs and AspRs are not separated by their racemase functions and form a serine/aspartate racemase family cluster based on phylogenetic analysis. Moreover, we have proposed that the AspR-specific triple serine loop region at amino acid positions 150-152 may be responsible for the large AspR activity. In the present study, to test this hypothesis, we prepared and characterized fourteen mutants in this region of animal SerRs and AspRs. The large AspR activity in Acropora and Crassostrea AspR was reduced to <0.04% of wild-type after substitution of the triple serine loop region. Conversely, introducing the triple serine loop region into Acropora, Crassostrea, and Penaeus SerR drastically increased the AspR activity. Those mutants showed similar or higher substrate affinity for aspartate than serine and showed 11-683-fold higher k cat and 28-351-fold higher k cat /K m values for aspartate than serine racemization. Furthermore, we introduced serine residues in all combinations at position 150-152 in mouse SerR. These mutants revealed that a change in the enzyme function from SerR to AspR can be caused by introduction of Ser151 and Ser152, and addition of the third serine residue at position 150 further enhances the enzyme specificity for aspartate due to a decrease in the serine racemase and serine dehydratase activity. Here, we provide convincing evidence that the AspR gene has evolved from the SerR gene by acquisition of the triple serine loop region.

Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral Capnella imbricata. Both enzymes initially catalyze the formation of 8R-hydroperoxy-eicosatetraenoic acid (8R-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in C. imbricata hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and C. imbricata cHPL. The amino acid replacements in cHPL-R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD-with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of P. homomalla cAOS with 8R-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8R-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.

Group I introns and homing endonuclease genes (HEGs) are mobile genetic elements, capable of invading target sequences in intron-less genomes. LAGLIDADG HEGs are the largest family of endonucleases, playing a key role in the mobility of group I introns in a process known as 'homing'. Group I introns and HEGs are rare in metazoans, and can be mainly found inserted in the COXI gene of some sponges and cnidarians, including stony corals (Scleractinia) and mushroom corals (Corallimorpharia). Vertical and horizontal intron transfer mechanisms have been proposed as explanations for intron occurrence in cnidarians. However, the central role of LAGLIDADG motifs in intron mobility mechanisms remains poorly understood. To resolve questions regarding the evolutionary origin and distribution of group I introns and HEGs in Scleractinia and Corallimorpharia, we examined intron/HEGs sequences within a comprehensive phylogenetic framework. Analyses of LAGLIDADG motif conservation showed a high degree of degradation in complex Scleractinia and Corallimorpharia. Moreover, the two motifs lack the respective acidic residues necessary for metal-ion binding and catalysis, potentially impairing horizontal intron mobility. In contrast, both motifs are highly conserved within robust Scleractinia, indicating a fully functional endonuclease capable of promoting horizontal intron transference. A higher rate of non-synonymous substitutions (Ka) detected in the HEGs of complex Scleractinia and Corallimorpharia suggests degradation of the HEG, whereas lower Ka rates in robust Scleractinia are consistent with a scenario of purifying selection. Molecular-clock analyses and ancestral inference of intron type indicated an earlier intron insertion in complex Scleractinia and Corallimorpharia in comparison to robust Scleractinia. These findings suggest that the lack of horizontal intron transfers in the former two groups is related to an age-dependent degradation of the endonuclease activity. Moreover, they also explain the peculiar geographical patterns of introns in stony and mushroom corals.

Healthy and diseased corals are threatened by different anthropogenic sources, such as pollution, a problem expected to become more severe in the near future. Despite the fact that coastal pollution and coral diseases might represent a serious threat to coral reef health, there is a paucity of controlled experiments showing whether the response of diseased and healthy corals to xenobiotics differs. In this study, we exposed healthy and Caribbean yellow band disease (CYBD)-affected Orbicella faveolata colonies to 3 sublethal concentrations of anthracene to test if enzymatic responses to this hydrocarbon were compromised in CYBD-affected tissues. For this, a 2-factorial fully orthogonal design was used in a controlled laboratory bioassay, using tissue condition (2 levels: apparently healthy and diseased) and pollutant concentration (4 levels: experimental control, 10, 30 and 100 ppb concentration) as fixed factors. A permutation-based ANOVA (PERMANOVA) was used to test the effects of condition and concentration on the specific activity of 3 enzymatic biomarkers: catalase, glutathione S-transferase, and glutathione peroxidase. We found a significant interaction between the concentration of anthracene and the colony condition for catalase (Pseudo-F = 3.84, df = 3, p < 0.05) and glutathione S-transferase (Pseudo-F = 3.29, df = 3, p < 0.05). Moreover, our results indicated that the enzymatic response to anthracene in CYBD-affected tissues was compromised, as the activity of these enzymes decreased 3- to 4-fold compared to healthy tissues. These results suggest that under a potential scenario of increasing hydrocarbon coastal pollution, colonies of O. faveolata affected with CYBD might become more vulnerable to the deleterious effects of chemical pollution.

The distribution of corals in Japan covers a wide range of latitudes, encompassing tropical to temperate zones. However, coral communities in temperate zones contain only a small subset of species. Among the parameters that determine the distribution of corals, temperature plays an important role. We tested the resilience to cold stress of three coral species belonging to the genus Acropora in incubation experiments. Acropora pruinosa, which is the northernmost of the three species, bleached at 13 °C, but recovered once temperatures were increased. The two other species, A. hyacinthus and A. solitaryensis, which has a more southerly range than A. pruinosa, died rapidly after bleaching at 13 °C. The physiological effects of cold bleaching on the corals included decreased rates of photosynthesis, respiration, and calcification, similar to the physiological effects observed with bleaching due to high temperature stress. Contrasting hot bleaching, no increases in antioxidant enzyme activities were observed, suggesting that reactive oxygen species play a less important role in bleaching under cold stress. These results confirmed the importance of resilience to cold stress in determining the distribution and northern limits of coral species, as cold events causing coral bleaching and high mortality occur regularly in temperate zones.

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π-cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π-cation bridge in 11R-LOX along with Arg106 and Asp173-a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Most animals rely on circulating hemocytes as cellular effectors of immunity. These cells traditionally have been characterized by morphology, function, and cellular contents. Morphological descriptions use granule differences and cell shapes; functional descriptions rely on phagocytic ability and oxygen transport; and cellular content descriptions include cytochemical features and key enzymes. Key enzymes used to identify phagocytes in tissues include hydrolytic enzymes, peroxidase, and--in invertebrates--phenoloxidase. Cnidaria such as Swiftia exserta lack a circulatory system, thereby complicating the identification of immune effector cells. As a first step in identifying immunocytes, this study focused on basic enzymes used during phagocytosis and encapsulation; both processes have been reported in octocorals such as S. exserta. Earlier work suggested that there are two populations of phagocytic cells: a constitutive population and an induced population following a trauma-associated challenge. To identify the constitutive immune effector cells in S. exserta in a nonactivated state, we used cryosections of unstimulated animals and the following enzymes to serve as identifying proxies due to their roles in phagocytosis and encapsulation: (1) acid phosphatase, (2) alkaline phosphatase, (3) non-specific esterase, (4) β-glucuronidase, (5) peroxidase, and (6) phenoloxidase. Our results indicate that in unstimulated animals, two distinct cell populations could function as immunocytes. These cell types were differentiated by their enzyme reactivity and their location within the mesoglea of S. exserta, and have been described as either "oblong granular cells" or "granular amoebocytes.", (© 2015 Marine Biological Laboratory.)

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using (18)O-labeled substrate and incubations in H2(18)O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Reef-building corals import inorganic carbon (Ci) to build their calcium carbonate skeletons and to support photosynthesis by the symbiotic algae that reside in their tissue. The internal pathways that deliver Ci for both photosynthesis and calcification are known to involve the enzyme carbonic anhydrase (CA), which interconverts CO2 and HCO3 (-). We have developed a method for absolute quantification of internal CA (iCA) activity in coral tissue based on the rate of (18)O-removal from labeled Ci. The method was applied to three Caribbean corals (Orbicella faveolata, Porites astreoides and Siderastrea radians) and showed that these species have similar iCA activities per unit surface area, but that S. radians has ∼10-fold higher iCA activity per unit tissue volume. A model of coral Ci processing shows that the measured iCA activity is sufficient to support the proposed roles for iCA in Ci transport for photosynthesis and calcification. This is the case even when iCA activity is homogeneously distributed throughout the coral, but the model indicates that it would be advantageous to concentrate iCA in the spaces where calcification (the calcifying fluid) and photosynthesis (the oral endoderm) take place. We argue that because the rates of photosynthesis and calcification per unit surface area are similar among the corals studied here, the areal iCA activity used to deliver Ci for these reactions should also be similar. The elevated iCA activity per unit volume of S. radians compared with that of the other species is probably due to the thinner effective tissue thickness in this species., (© 2015. Published by The Company of Biologists Ltd.)

In this chapter, we discuss the influence of an anisotropic protein environment on the reaction mechanisms of saccharopine reductase and uroporphyrinogen decarboxylase, respectively, via the use of a quantum mechanical and molecular mechanical (QM/MM) approach. In addition, we discuss the importance of selecting a suitable DFT functional to be used in a QM/MM study of a key intermediate in the mechanism of 8R-lipoxygenase, a nonheme iron enzyme. In the case of saccharopine reductase, while the enzyme utilizes a substrate-assisted catalytic pathway, it was found that only through treating the polarizing effect of the active site, via the use of an electronic embedding formalism, was agreement with experimental kinetic data obtained. Similarly, in the case of uroporphyrinogen decarboxylase, the effect of the protein environment on the catalytic mechanism was found to be such that the calculated rate-limiting barrier is in good agreement with related experimentally determined values for the first decarboxylation of the substrate. For 8R-lipoxygenase, it was found that the geometries and energies of the multicentered open-shell intermediate complexes formed during the mechanism are quite sensitive to the choice of the density functional theory method. Thus, while density functional theory has become the method of choice in QM/MM studies, care must be taken in the selection of a particular high-level method., (© 2015 Elsevier Inc. All rights reserved.)

Reef-building corals switch endosymbiotic algae of the genus Symbiodinium during their early growth stages and during bleaching events. Clade C Symbiodinium algae are dominant in corals, although other clades - including A and D - have also been commonly detected in juvenile Acroporid corals. Previous studies have been reported that only molecular data of Symbiodinium clade were identified within field corals. In this study, we inoculated aposymbiotic juvenile polyps with cultures of clades C1 and D Symbiodinium algae, and investigated the different effect of these two clades of Symbiodinium on juvenile polyps. Our results showed that clade C1 algae did not grow, while clade D algae grew rapidly during the first 2 months after inoculation. Polyps associated with clade C1 algae exhibited bright green fluorescence across the body and tentacles after inoculation. The growth rate of polyp skeletons was lower in polyps associated with clade C1 algae than those associated with clade D algae. On the other hand, antioxidant activity (catalase) of corals was not significantly different between corals with clade C1 and clade D algae. Our results suggested that clade D Symbiodinium algae easily form symbiotic relationships with corals and that these algae could contribute to coral growth in early symbiosis stages.

In octocorals, a catalase-like allene oxide synthase (AOS) and an 8R-lipoxygenase (LOX) gene are fused together encoding for a single AOS-LOX fusion protein. Although the AOS-LOX pathway is central to the arachidonate metabolism in corals, its biological function in coral homeostasis is unclear. Using an acute incision wound model in the soft coral Capnella imbricata, we here test whether LOX pathway, similar to its role in plants, can contribute to the coral damage response and regeneration. Analysis of metabolites formed from exogenous arachidonate before and after fixed time intervals following wounding indicated a significant increase in AOS-LOX activity in response to mechanical injury. Two AOS-LOX isoforms, AOS-LOXa and AOS-LOXb, were cloned and expressed in bacterial expression system as active fusion proteins. Transcription levels of corresponding genes were measured in normal and stressed coral by qPCR. After wounding, AOS-LOXa was markedly up-regulated in both, the tissue adjacent to the incision and distal parts of a coral colony (with the maximum reached at 1 h and 6 h post wounding, respectively), while AOS-LOXb was stable. According to mRNA expression analysis, combined with detection of eicosanoid product formation for the first time, the AOS-LOX was identified as an early stress response gene which is induced by mechanical injury in coral.

The health and existence of coral reefs are in danger by an increasing range of environmental and anthropogenic impacts. The causes of coral reef decline include worldwide climate change, shoreline development, habitat destruction, pollution, sedimentation and overexploitation. These disasters have contributed to an estimated loss of 27% of the reefs. If the current pressure continues unabated, the estimated loss of coral reef will be about 60% by the year 2030. Therefore, the present study was aimed to analyze the enzymes involved in stress induced by coral pathogen and its resistance. We focused on the enzymes involved in melanin synthesis pathway (phenoloxidase (PO) and peroxidases (POD)) and free radical scavenging enzymes (super oxide dismutase (SOD), catalase (CAT)) and glutathione peroxidase (Gpx) in selected scleractinian corals such as Acropora formosa, Echinopora lamellosa, Favia favus, Favites halicora, Porites sp., and Anacropora forbesi. Overall, PO activity of coral was significantly lower than that of zooxanthellae except for Favia favus. Coral colonies with lower PO and POD activities are prone to disease. Maximum antioxidant defensive enzymes were observed in Favia favus followed by Echinopora lamellose. It is concluded that assay of these enzymes can be used as biomarkers for identifying the susceptibility of corals towards coral bleaching induced by pathogen.

Coral allene oxide synthase (cAOS) catalyzes the formation of allene oxides from fatty acid hydroperoxides. Interestingly, its active site differs from that of catalase by only a single residue yet is incapable of catalase activity. That is, it is unable to catalyze the decomposition of hydrogen peroxide to molecular oxygen and water. However, the single active-site mutation T66V allows cAOS to exhibit catalase activity. We have performed a series of molecular dynamics (MD) simulations in order to gain insights into the differences in substrate (8R-hydroperoxyeicosatetraenoic) and H2O2 active site binding between wild-type cAOS and the T66V mutant cAOS. It is observed that in wild-type cAOS the active site Thr66 residue consistently forms a strong hydrogen-bonding interaction with H2O2 (catalase substrate) and, importantly, with the aid of His67 helps to pull H2O2 away from the heme Fe center. In contrast, in the T66V-cAOS mutant the H2O2 is much closer to the heme's Fe center and now forms a consistent Fe···O2H2 interaction. In addition, the His67···H2O2 distance shortens considerably, increasing the likelihood of a Cpd I intermediate and hence exhibiting catalase activity.

Protein engineering has created a palette of monomeric fluorescent proteins (FPs), but there remains an ~30 nm spectral gap between the most red-shifted useful Aequorea victoria green FP (GFP) variants and the most blue-shifted useful Discosoma sp. red FP (RFP) variants. To fill this gap, we have engineered a monomeric version of the yellow FP (YFP) from Zoanthus sp. coral. Our preferred variant, designated as mPapaya1, displays excellent fluorescent brightness, good photostability, and retains its monomeric character both in vitro and in living cells in the context of protein chimeras. We demonstrate that mPapaya1 can serve as a good Förster resonance energy transfer (FRET) acceptor when paired with an mTFP1 donor. mPapaya1 is a valuable addition to the palette of FP variants that are useful for multicolor imaging and FRET-based biosensing., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fluorescent proteins are used as noninvasive tags for protein trafficking, structure, and action. In this issue of Chemistry & Biology, Hoi and colleagues present a new optimized zFP538 yellow fluorescent protein called mPapaya1 that is bright, monomeric, and an excellent fusion partner for cellular proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The trimethylsilyl (TMS) peroxides of linoleic acid 9(S)-hydroperoxide (TMS or Me esters) were subjected to gas chromatography-mass spectrometry (GC-MS) analyses. The cyclopentenones, trans- and cis-10-oxo-11-phytoenoic acid (10-oxo-PEA, Me or TMS esters) were first time detected as the products of TMS-peroxide thermal conversions. The major products were ketodienes, epoxyalcohols, hemiacetals and decadienals. For further study of thermal cyclopentenone formation, 9(S)- or 13(S)-hydroperoxides of linoleic acid (Me esters) were sealed in ampoules and heated at 230 °C for 15 or 30 min. The products were separated by HPLC. The cyclopentenone fractions were collected and analyzed by GC-MS. Trans-10-oxo-PEA (Me) and 10-oxo-9(13)-PEA (Me) were formed during the thermal conversion of 9-hydroperoxide (Me ester). Similarly, the cyclopentenones trans-12-oxo-PEA (Me) and 12-oxo-9(13)-PEA (Me) were detected after the heating of 13-hydroperoxide (Me ester). Thermal formation of cyclopentenones can be considered as a biomimetic model of AOS pathway, providing new insights into the mechanisms of allene oxide formation and cyclization., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

One of the proposed pathways to the synthesis of 4-hydroxy-nonenal (HNE) and related aldehydes entails formation of an intermediate bis-allylic fatty acid dihydroperoxide. As a first direct demonstration of such a pathway and proof of principle, herein we show that 8R-lipoxygenase (8R-LOX) catalyzes the enzymatic production of the HNE-like product (11-oxo-8-hydroperoxy-undeca-5,9-dienoic acid) via synthesis of 8,11-dihydroperoxy-eicosa-5,9,12,14-tetraenoic acid intermediate. Incubation of arachidonic acid with 8R-LOX formed initially 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE), which was further converted to a mixture of products including a prominent HPNE-like enone. A new bis-allylic dihydroperoxide was trapped when the incubation was repeated on ice. Reincubation of this intermediate with 8R-LOX successfully demonstrated its conversion to the enone products, and this reaction was greatly accelerated by coincubation with NDGA, a reductant of the LOX iron. These findings identify a plausible mechanism that could contribute to the production of 4-hydroxy-alkenals in vivo.

Background: As a step towards understanding coral immunity we present the first whole transcriptome analysis of the acute responses of Acropora millepora to challenge with the bacterial cell wall derivative MDP and the viral mimic poly I:C, defined immunogens provoking distinct but well characterised responses in higher animals., Results: These experiments reveal similarities with the responses both of arthropods and mammals, as well as coral-specific effects. The most surprising finding was that MDP specifically induced three members of the GiMAP gene family, which has been implicated in immunity in mammals but is absent from Drosophila and Caenorhabditis. Like their mammalian homologs, GiMAP genes are arranged in a tandem cluster in the coral genome., Conclusions: A phylogenomic survey of this gene family implies ancient origins, multiple independent losses and lineage-specific expansions during animal evolution. Whilst functional convergence cannot be ruled out, GiMAP expression in corals may reflect an ancestral role in immunity, perhaps in phagolysosomal processing.

In this present work the mechanism by which cAOS catalyzes the formation of allene oxide from its hydroperoxy substrate was computationally investigated by using a DFT-chemical cluster approach. In particular, the effects of dispersion interactions and DFT functional choice (M06, B3LYP, B3LYP*, and BP86), as well as the roles of multistate reactivity and the tyrosyl proximal ligand, were examined. It is observed that the computed relative free energies of stationary points along the overall pathway are sensitive to the choice of DFT functional, while the mechanism obtained is generally not. Large reductions in relative free energies for stationary points along the pathway (compared to the initial reactant complex) of on average 46.3 and 97.3 kJ mol(-1) for the doublet and quartet states, respectively, are observed upon going from the M06 to BP86 functional. From results obtained by using the B3LYP* method, well-tested previously on heme-containing systems, the mechanism of cAOS appears to occur with considerably higher Gibbs free energies than that for the analogous pathway in pAOS, possibly due to the presence of a ligating tyrosyl residue in cAOS. Furthermore, at the IEFPCM-B3LYP*/6-311+G(2df,p)//B3LYP/BS1 level of theory the inclusion of dispersion effects leads to the suggestion that the overall mechanism of cAOS could occur without the need for spin inversion.

Scleractinian coral stylophora pistillata carbonic anhydrase (STPCA) enzyme is a secreted isoform, plays a direct role in bio-mineralization. Sulfonamides, including some clinically used derivatives are the most important class of STPCA inhibitors. In order to search for efficient STPCA inhibitors molecules, the present work deals with quantitative structure-activity relationship (QSAR) studies of a series of 36 bioactive molecules. A heuristic algorithm selects the best multiple linear regression (MLR) equation showed the correlation between the observed values and the calculated values of activity is very good (N=36, Se=0.1683, r(2)=0.9158, F=54.3809, rcv(2)=0.8569). The novelty of this work is not only to explore the structural attributes of bioactive molecules but also to design and predict in silico the STPCA inhibitory activity of new not yet synthesized compounds. The analyzed prediction set includes many molecules having greater computed activity than observed value of inhibitory activity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Coral reefs are among the most biologically diverse and economically important ecosystems on the planet. The deposition of massive calcium carbonate skeletons (biomineralization or calcification) by scleractinian corals forms the coral reef framework/architecture that serves as habitat for a large diversity of organisms. This process would not be possible without the intimate symbiosis between corals and photosynthetic dinoflagellates, commonly called zooxanthellae. Carbonic anhydrases play major roles in those two essential processes of coral's physiology: they are involved in the carbon supply for calcium carbonate precipitation as well as in carbon-concentrating mechanisms for symbiont photosynthesis. Here, we review the current understanding of diversity and function of carbonic anhydrases in corals and discuss the perspective of theses enzymes as a key to understanding impacts of environmental changes on coral reefs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lipoxygenases (LOXs) are ubiquitous in nature and catalyze a range of life-essential reactions within organisms. In particular they are critical to the formation of eicosanoids, which are critical for normal cell function. However, a number of important questions about the reactivity and mechanism of these enzymes still remain. Specifically, although the initial step in the mechanism of LOXs has been well studied, little is known of subsequent steps. Thus, with use of a quantum mechanical/molecular mechanical approach, the complete catalytic mechanism of (8R)-LOX was investigated. The results have provided a better understanding of the general chemistry of LOXs as a whole. In particular, from comparisons with soybean LOX-1, it appears that the initial proton-coupled electron transfer may be very similar among all LOXs. Furthermore, LOXs appear to undergo multistate reactivity where potential spin inversion of an electron may occur either in the attack of O(2) or in the regeneration of the active site. Lastly, it is shown that with the explicit modeling of the environment, the regeneration of the active center likely occurs via the rotation of the intermediate followed by an outer-sphere [Formula: see text] transfer as opposed to the formation of a "purple intermediate" complex.

The performance of a range density functional theory functionals combined in a quantum mechanical (QM)/molecular mechanical (MM) approach was investigated in their ability to reliably provide geometries, electronic distributions, and relative energies of a multicentered open-shell mechanistic intermediate in the mechanism 8R-Lipoxygenase. With the use of large QM/MM active site chemical models, the smallest average differences in geometries between the catalytically relevant quartet and sextet complexes were obtained with the B3LYP(*) functional. Moreover, in the case of the relative energies between (4) II and (6) II, the use of the B3LYP(*) functional provided a difference of 0.0 kcal mol(-1). However, B3LYP(±) and B3LYP also predicted differences in energies of less than 1 kcal mol(-1). In the case of describing the electronic distribution (i.e., spin density), the B3LYP(*), B3LYP, or M06-L functionals appeared to be the most suitable. Overall, the results obtained suggest that for systems with multiple centers having unpaired electrons, the B3LYP(*) appears most well rounded to provide reliable geometries, electronic structures, and relative energies., (Copyright © 2012 Wiley Periodicals, Inc.)

Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had >1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels.

Secreted peptides, produced by enzymatic processing of larger precursor molecules, are found throughout the animal kingdom and play important regulatory roles as neurotransmitters and hormones. Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity. Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL). In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification. Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. Here, we report sequence and expression data for two PAMs from the coral Acropora millepora (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. These analyses indicate that bifunctional PAMs predate the origins of the nervous and endocrine systems, consistent with the idea that within the Metazoa their ancestral function may have been to amidate epitheliopeptides. More surprisingly, the phylogenomic survey also revealed the presence of PAMs in green algae (but not in higher plants or fungi), implying that the bifunctional enzyme either predates the plant/animal divergence and has subsequently been lost in a number of lineages or perhaps that convergent evolution or lateral gene transfer has occurred. This finding is consistent with recent discoveries that other molecules once thought of as "neural" predate nervous systems.

Coral reefs are threatened by increasing levels of coral disease and the functional loss of obligate algal symbionts (bleaching). Levels of immunity relate directly to susceptibility to these threats; however, our understanding of fundamental aspects of coral immunology is lacking. We show that three melanin-synthesis pathway components (mono-phenoloxidase, ortho-diphenoloxidase (tyrosinase-type pathway) and para-diphenoloxidase (laccase-type pathway)) are present in both their active (phenoloxidase, PO) and inactive (prophenoloxidase, PPO) forms across a diverse range of 22 species of healthy Indo-Pacific anthozoans. We also demonstrate transglutaminase activity of the coagulation cascade for, to our knowledge, the first time in a coral. Melanin-synthesis enzyme activities varied among taxa, although they were generally lowest in the coral family Acroporidae and highest in the Poritidae and Oculinidae. Inactive tyrosinase-type activity (PPO) and active laccase-type activity (PO) correlate with taxonomic patterns in disease resistance, whereas the converse pattern in activity levels correlates with bleaching resistance. Overall, we demonstrate the presence of several melanin-synthesis pathways in Indo-Pacific corals, co-regulation among some pathway components, and highlight their potential roles in coral health.

Conversion of fatty acid hydroperoxides to epoxyalcohols is a well known secondary reaction of lipoxygenases, described for S-specific lipoxygenases forming epoxyalcohols with a trans-epoxide configuration. Here we report on R-specific lipoxygenase synthesis of a cis-epoxyalcohol. Although arachidonic and dihomo-γ-linolenic acids are metabolized by extracts of the Caribbean coral Plexaura homomalla via 8R-lipoxygenase and allene oxide synthase activities, 20:3ω6 forms an additional prominent product, identified using UV, GC-MS, and NMR in comparison to synthetic standards as 8R,9S-cis-epoxy-10S-erythro-hydroxy-eicosa-11Z,14Z-dienoic acid. Both oxygens of (18)O-labeled 8R-hydroperoxide are retained in the product, indicating a hydroperoxide isomerase activity. Recombinant allene oxide synthase formed only allene epoxide from 8R-hydroperoxy-20:3ω6, whereas two different 8R-lipoxygenases selectively produced the epoxyalcohol.A biosynthetic scheme is proposed in which a partial rotation of the reacting intermediate is required to give the observed erythro epoxyalcohol product. This characteristic and the synthesis of cis-epoxy epoxyalcohol may be a feature of R-specific lipoxygenases.

Benthic macroalgae can be abundant on present-day coral reefs, especially where rates of herbivory are low and/or dissolved nutrients are high. This study investigated the impact of macroalgal extracts on both coral-associated bacterial assemblages and sublethal stress response of corals. Crude extracts and live algal thalli from common Caribbean macroalgae were applied onto the surface of Montastraea faveolata and Porites astreoides corals on reefs in both Florida and Belize. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons was used to examine changes in the surface mucus layer (SML) bacteria in both coral species. Some of the extracts and live algae induced detectable shifts in coral-associated bacterial assemblages. However, one aqueous extract caused the bacterial assemblages to shift to an entirely new state (Lobophora variegata), whereas other organic extracts had little to no impact (e.g. Dictyota sp.). Macroalgal extracts more frequently induced sublethal stress responses in M. faveolata than in P. astreoides corals, suggesting that cellular integrity can be negatively impacted in selected corals when comparing co-occurring species. As modern reefs experience phase-shifts to a higher abundance of macroalgae with potent chemical defenses, these macroalgae are likely impacting the composition of microbial assemblages associated with corals and affecting overall reef health in unpredicted and unprecedented ways.

Disease epizootics are increasing with climatic shifts, yet within each system only a subset of species are identified as the most vulnerable. Understanding ecological immunology patterns as well as environmental influences on immune defenses will provide insight into the persistence of a functional system through adverse conditions. Amongst the most threatened ecosystems are coral reefs, with coral disease epizootics and thermal stress jeopardizing their survival. Immune defenses were investigated within three Caribbean corals, Montastraea faveolata, Stephanocoenia intersepta and Porites astreoides, which represent a range of disease and bleaching susceptibilities. Levels of several immune parameters were measured in response to elevated water temperature and the presence of a commercial pathogen-associated molecular pattern (PAMP) - lipopolysaccharide (LPS) - as an elicitor of the innate immune response. Immune parameters included prophenoloxidase (PPO) activity, melanin concentration, bactericidal activity, the antioxidants peroxidase and catalase, and fluorescent protein (FP) concentration. LPS induced an immune response in all three corals, although each species responded differently to the experimental treatments. For example, M. faveolata, a disease-susceptible species, experienced significant decreases in bactericidal activity and melanin concentration after exposure to LPS and elevated temperature alone. Porites astreoides, a disease-resistant species, showed increased levels of enzymatic antioxidants upon exposure to LPS independently and increased PPO activity in response to the combination of LPS and elevated water temperature. This study demonstrates the ability of reef-building corals to induce immune responses in the presence of PAMPs, indicating activation of PAMP receptors and the transduction of appropriate signals leading to immune effector responses. Furthermore, these data address the emerging field of ecological immunology by highlighting interspecific differences in immunity and immunocompetences among Caribbean corals, which are reflected in their life-history characteristics, disease susceptibilities and bleaching-induced mortality.

Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (<20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is a putative organic peroxide-dependent peroxidase. To elucidate factors influencing the functions of these and related heme proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme enzymes in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg(+)-N(ω)-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O(2) states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg(+)-N(ω)-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC., (Copyright © 2011 Elsevier Inc. All rights reserved.)