Application of MALDI-MS for characterization of fucoidan hydrolysates and screening of endo-fucoidanase activity.

Brown macroalgae synthesize large amounts of fucoidans, sulfated fucose-containing polysaccharides, in the ocean. Fucoidans are of importance for their recently discovered contribution to marine carbon dioxide sequestration and due to their potential applications in biotechnology and biomedicine. However, fucoidans have high intra- and intermolecular diversity that challenges assignment of structure to biological function and the development of applications. Fucoidan-active enzymes may be used to simplify this diversity by producing defined oligosaccharides more applicable for structural refinement, characterization, and structure to function assignment for example via bioassays. In this study, we combined MALDI mass spectrometry with biocatalysis to show that the endo-fucoidanases P5AFcnA and Wv323 can produce defined oligosaccharide structures directly from unrefined macroalgal biomass. P5AFcnA released oligosaccharides from seven commercial fucoidan extracts in addition to unrefined biomass of three macroalgae species indicating a broadly applicable approach reproducible across 10 species. Both MALDI-TOF/TOF and AP-MALDI-Orbitrap systems were used, demonstrating that the approach is not instrument-specific and exploiting their combined high-throughput and high-resolution capabilities. Overall, the combination of MALDI-MS and endo-fucoidanase assays offers high-throughput evaluation of fucoidan samples and also enables extraction of defined oligosaccharides of known structure from unrefined seaweed biomass.

Functional identification of the 4-deoxy-L-erythro-5-hexoseulose uronate reductase from a brown alga, Saccharina japonica.

We previously reported the alginate lyase, SjAly, from a brown alga, Saccharina japonica, providing the first experimental evidence for a functional alginate-degradation enzyme in brown algae. 4-deoxy-L-erythro-5-hexoseulose uronate (DEHU), derived from an unsaturated monosaccharide, was identified as the minimum degradation product produced by SjAly-mediated lysis of alginate. DEHU was hitherto reported to be reduced to 2-keto-3-deoxy-gluconate (KDG) by a DEHU-specific reductase with NAD(P)H in alginate-assimilating organisms and its metabolism in alginate-producing organisms is unknown. Here, we report the functional identification of a DEHU reductase, SjRed, in S. japonica. Among the 14 tested compounds, only DEHU was used as a substrate and was converted to KDG in the presence of NADPH. Optimum temperature, pH, and KCl concentration required for SjRed activity were determined to be 25 °C, 7.2, and 100 mM, respectively. SjRed consists of 341 amino acid residues and is proposed to be a member of the aldo-keto reductase superfamily. Sequencing of SjRed revealed that it is composed of at least three exons. These results indicate the existence of an enzyme that reduces DEHU to KDG in S. japonica. This is the first report on the functional identification of a DEHU-reductase in alginate-producing organisms.

Properties and potential applications of mannuronan C5-epimerase: A biotechnological tool for modifying alginate.

Given the excellent characteristics of alginate, it is an industrially important polysaccharide. Mannuronan C5-epimerase (MC5E) is an alginate-modifying enzyme that catalyzes the conversion of ß-D-mannuronate (M) to its C5 epimer α-L-guluronate (G) in alginate. Both the biological activities and physical properties of alginate are determined by M/G ratios and distribution patterns. Therefore, MC5E is regarded as a biotechnological tool for modifying and processing alginate. Various MC5Es derived from brown algae, Pseudomonas and Azotobacter have been isolated and characterized. With the rapid development of structural biology, the crystal structures and catalytic mechanisms of several MC5Es have been elucidated. It is necessary to comprehensively understand the research status of this alginate-modifying enzyme. In this review, the properties and potential applications of MC5Es isolated from different kinds of organisms are summarized and reviewed. Moreover, future research directions of MC5Es as well as strategies to enhance their properties are elucidated, highlighted, and prospected.

Exploiting Mannuronan C-5 Epimerases in Commercial Alginate Production.

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae (Ascophyllum nodosum, Durvillea potatorum, Laminaria hyperborea and Lessonia nigrescens) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance (1H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

Characteristics and applications of alginate lyases: A review.

Brown algae, as the main source of alginate, are a type of marine biomass with a very high output. Alginate, a polysaccharide composed of ß-D-mannuronic acid (M) and α-L-guluronic acid (G), has great potential for applications in the food, cosmetic and pharmaceutical industries. Alginate lyases (Alys) can degrade alginate polymers into oligosaccharides or monosaccharides, resulting in a broad application field. Alys can be used for both the production of alginate oligosaccharides and the biorefinery of brown algae. In view of their important functions, an increasing number of Alys have been isolated and characterized. For better application, a comprehensive understanding of Alys is essential. Therefore, in this paper, we summarized recently discovered Alys, discussed their characteristics, and introduced their structural properties, degradation patterns and biological roles in alginate-degrading organisms. In addition, applications of Alys have been illustrated with examples. This paper provides a relatively comprehensive description of Alys, which is significant for Alys exploration and application.

Genome-wide analysis of the Saccharina japonica sulfotransferase genes and their transcriptional profiles during whole developmental periods and under abiotic stresses.

BACKGROUND: As a unique sulfated polysaccharide, fucoidan is an important component of cell wall in brown seaweeds. Its biochemical properties are determined by the positions and quantity of sulfate groups. Sulfotransferases (STs) catalyze the sulfation process, which transfer the sulfuryl groups to carbohydrate backbones and are crucial for fucoidan biosynthesis. Nevertheless, the structures and functions of STs in brown seaweeds are rarely investigated. RESULTS: There are a total of 44 ST genes identified from our genome and transcriptome analysis of Saccharina japonica, which were located in the 17 scaffolds and 11 contigs. The S. japonica ST genes have abundant introns and alternative splicing sites, and five tandem duplicated gene clusters were identified. Generally, the ST genes could be classified into five groups (Group I ~ V) based on phylogenetic analysis. Accordingly, the ST proteins, which were encoded by genes within the same group, contained similar conserved motifs. Members of the S. japonica ST gene family show various expression patterns in different tissues and developmental stages. Transcriptional profiles indicate that the transcriptional levels of more than half of the ST genes are higher in kelp basal blades than in distal blades. Except for ST5 and ST28, most ST genes are down-regulated with the kelp development stages. The expression levels of nine ST genes were detected by real-time quantitative PCR, which demonstrates that they responded to low salinity and drought stresses. CONCLUSIONS: Various characteristics of the STs allow the feasibilities of S. japonica to synthesize fucoidans with different sulfate groups. This enables the kelp the potential to adapt to the costal environments and meet the needs of S. japonica growth.

An algal enzyme required for biosynthesis of the most abundant marine carotenoids.

Fucoxanthin and its derivatives are the main light-harvesting pigments in the photosynthetic apparatus of many chromalveolate algae and represent the most abundant carotenoids in the world's oceans, thus being major facilitators of marine primary production. A central step in fucoxanthin biosynthesis that has been elusive so far is the conversion of violaxanthin to neoxanthin. Here, we show that in chromalveolates, this reaction is catalyzed by violaxanthin de-epoxidase-like (VDL) proteins and that VDL is also involved in the formation of other light-harvesting carotenoids such as peridinin or vaucheriaxanthin. VDL is closely related to the photoprotective enzyme violaxanthin de-epoxidase that operates in plants and most algae, revealing that in major phyla of marine algae, an ancient gene duplication triggered the evolution of carotenoid functions beyond photoprotection toward light harvesting.

Identification, classification, and evolution of putative xylosyltransferases from algae.

Xylosyltransferases (XylTs) play key roles in the biosynthesis of many different polysaccharides. These enzymes transfer D-xylose from UDP-xylose to substrate acceptors. In this study, we identified 30 XylTs from primary endosymbionts (green algae, red algae, and glaucophytes) and secondary or higher endosymbionts (brown algae, diatoms, Eustigmatophyceae, Pelagophyceae, and Cryptophyta). We performed comparative phylogenetic studies on key XylT subfamilies, and investigated the functional divergence of genes using RNA-Seq. Of the 30 XylTs, one ß-1,4-XylT IRX14-related, one ß-1,4 XylT IRX10L-related, and one xyloglucan 6-XylT 1-related gene were identified in the Charophyta, showing strong similarities to their land plant descendants. This implied the ancient occurrence of xylan and xyloglucan biosynthetic machineries in Charophyta. The other 27 XylTs were identified as UDP-D-xylose: L-fucose-α-1,3-D-XylT (FucXylT) type that specifically transferred D-xylose to fucose. We propose that FucXylTs originated from the last eukaryotic common ancestor, rather than being plant specific, because they are also distributed in Choanoflagellatea and Echinodermata. Considering the evidence from many aspects, we hypothesize that the FucXylTs likely participated in fucoidan biosynthesis in brown algae. We provide the first insights into the evolutionary history and functional divergence of FucXylT in algal biology.

Diversity and evolution of cytochromes P450 in stramenopiles.

MAIN CONCLUSION: Comparative genomic analysis of cytochromes P450 revealed high diversification and dynamic changes in stramenopiles, associated with transcriptional responsiveness to various environmental stimuli. Comparative genomic and molecular evolution approaches were used to characterize cytochromes P450 (P450) diversity in stramenopiles. Phylogenetic analysis pointed to a high diversity of P450 in stramenopiles and identified three major clans. The CYP51 and CYP97 clans were present in brown algae, diatoms and Nannochloropsis gaditana, whereas the CYP5014 clan mainly includes oomycetes. Gene gain and loss patterns revealed that six CYP families-CYP51, CYP97, CYP5160, CYP5021, CYP5022, and CYP5165-predated the split of brown algae and diatoms. After they diverged, diatoms gained more CYP families, especially in the cold-adapted species Fragilariopsis cylindrus, in which eight new CYP families were found. Selection analysis revealed that the expanded CYP51 family in the brown alga Cladosiphon okamuranus exhibited a more relaxed selection constraint compared with those of other brown algae and diatoms. Our RNA-seq data further evidenced that most of P450s in Saccharina japonica are highly expressed in large sporophytes, which could potentially promote the large kelp formation in this developmental stage. A survey of Ectocarpus siliculosus and diatom transcriptomes showed that many P450s are responsive to stress, nutrient limitation or light quality, suggesting pivotal roles in detoxification or metabolic processes under adverse environmental conditions. The information provided in this study will be helpful in designing functional experiments and interpreting P450 roles in this particular lineage.

Discovery and screening of novel metagenome-derived GH107 enzymes targeting sulfated fucans from brown algae.

Sulfated fucans, often denoted as fucoidans, are highly variable cell wall polysaccharides of brown algae, which possess a wide range of bioactive properties with potential pharmaceutical applications. Due to their complex architecture, the structures of algal fucans have until now only been partly determined. Enzymes capable of hydrolyzing sulfated fucans may allow specific release of defined bioactive oligosaccharides and may serve as a tool for structural elucidation of algal walls. Currently, such enzymes include only a few hydrolases belonging to the glycoside hydrolase family 107 (GH107), and little is known about their mechanistics and the substrates they degrade. In this study, we report the identification and recombinant production of three novel GH107 family proteins derived from a marine metagenome. Activity screening against a large substrate collection showed that all three enzymes degraded sulfated fucans from brown algae in the order Fucales. This is in accordance with a hydrolytic activity against α-1,4-fucosidic linkages in sulfated fucans as reported for previous GH107 members. Also, the activity screening gave new indications about the structural differences in brown algal cell walls. Finally, sequence analyses allowed identification of the proposed catalytic residues of the GH107 family. The findings presented here form a new basis for understanding the GH107 family of enzymes and investigating the complex sulfated fucans from brown algae. DATABASE: The assembled metagenome and raw sequence data is available at EMBL-EBI (Study number: PRJEB28480). Sequences of the GH107 fucanases (Fp273, Fp277, and Fp279) have been deposited in GenBank under accessions MH755451-MH755453.

Characterization of a new endo-type polysaccharide lyase (PL) family 6 alginate lyase with cold-adapted and metal ions-resisted property.

Alginate lyase played an important role in brown algae degradation, and its enzymatic degradation products showed various biological activities. Although many alginate lyases have been characterized, the enzymes with special characterizations are still rather rare. In this study, a new alginate lyase gene, tsaly6A, has been cloned from marine bacterium Thalassomonas sp. LD5, and expressed in Escherichia coli. The deduced alginate lyase, TsAly6A, belonged to the polysaccharide lyase (PL) family 6 and showed the highest amino acid identity (63%) with an exo-type oligoalginate lyase AlyGC. However, this study showed that TsAly6A was an endo-type enzyme yielding alginate trisaccharides (64.5%) as the main products. Compared with other alginate lyases, TsAly6A showed high trisaccharide-yielding levels. Meanwhile, TsAly6A showed the specific activity of 15,960 U/µmol at its optimal pH (pH 8.0) and temperature (35 °C). In addition, TsAly6A was a cold-adapted, salt-activated and metal ions-resisted alginate lyase, which will enable it to perform high activity in the solution containing various ions. Its cold-adaptation, metal ions-tolerance and high trisaccharides yields make TsAly6A an excellent candidate for industrial applications.

Analyzing Rho GTPase-Dependent Processes During Cell Polarization in Brown Algae.

Zygotes of the fucoid brown algae are useful models for investigating the molecular and cellular mechanisms of cell polarization. These organisms are abundant in the marine intertidal zone, where they grow firmly anchored to rocks. In response to environmental cues like sunlight, zygotes generate asymmetries within the cell that ultimately establish an axis of growth. The transduction of these cues relies on Rac1-mediated signaling that remodels the actin cytoskeleton, alters patterns of endocytosis and secretion, and ultimately prepares the zygote for localized (tip) growth. This chapter presents protocols for obtaining synchronous populations of zygotes, and for detecting changes in filamentous actin arrays, endomembrane patterns, and secretion patterns that occur during light-induced polarization.

Biotechnological Applications of Marine Enzymes From Algae, Bacteria, Fungi, and Sponges.

Diversity is the hallmark of all life forms that inhabit the soil, air, water, and land. All these habitats pose their unique inherent challenges so as to breed the "fittest" creatures. Similarly, the biodiversity from the marine ecosystem has evolved unique properties due to challenging environment. These challenges include permafrost regions to hydrothermal vents, oceanic trenches to abyssal plains, fluctuating saline conditions, pH, temperature, light, atmospheric pressure, and the availability of nutrients. Oceans occupy 75% of the earth's surface and harbor most ancient and diverse forms of organisms (algae, bacteria, fungi, sponges, etc.), serving as an excellent source of natural bioactive molecules, novel therapeutic compounds, and enzymes. In this chapter, we introduce enzyme technology, its current state of the art, unique enzyme properties, and the biocatalytic potential of marine algal, bacterial, fungal, and sponge enzymes that have indeed boosted the Marine Biotechnology Industry. Researchers began exploring marine enzymes, and today they are preferred over the chemical catalysts for biotechnological applications and functions, encompassing various sectors, namely, domestic, industrial, commercial, and healthcare. Next, we summarize the plausible pros and cons: the challenges encountered in the process of discovery of the potent compounds and bioactive metabolites such as biocatalysts/enzymes of biomedical, therapeutic, biotechnological, and industrial significance. The field of Marine Enzyme Technology has recently assumed importance, and if it receives further boost, it could successfully substitute other chemical sources of enzymes useful for industrial and commercial purposes and may prove as a beneficial and ecofriendly option. With appropriate directions and encouragement, marine enzyme technology can sustain the rising demand for enzyme production while maintaining the ecological balance, provided any undesired exploitation of the marine ecosystem is avoided.

Usefulness of Alginate Lyases Derived from Marine Organisms for the Preparation of Alginate Oligomers with Various Bioactivities.

Alginate-degrading enzyme, alginate lyase, catalyzes the cleavage of glycosidic 1-4 O-linkages between uronic acid residues of alginate by a ß-elimination reaction leaving a 4-deoxy-l-erythro-hex-4-ene pyranosyluronate as nonreducing terminal end. The enzymes from a wide variety of sources such as marine molluscs, seaweeds, and marine bacteria have been discovered and studied not only from a point of view of enzymological interest of enzyme itself but also for elucidation of fine chemical structure of alginate, structure-activity relationship of alginate, and biological activities and physicochemical features of the enzymatic digestion products. Based on the substrate specificities, alginate lyases are classified into three groups: poly(ß-d-mannuronate) lyase, poly(α-l-guluronate) lyase, and bifunctional alginate lyase, which are specific to mannuronate, guluronate, and both uronic acid residues, respectively. We have studied enzymological aspects of these three types of alginate lyases, and bioactivities of enzymatically digested alginate oligomers. In this chapter, we described the purification and characterization of three types of alginate lyases from different marine origins and overviewed the bioactivities of alginate oligomers.

Functional Exploration of the Polysaccharide Lyase Family PL6.

Alginate, the main cell-wall polysaccharide of brown algae, is composed of two residues: mannuronic acid (M-residues) and, its C5-epimer, guluronic acid (G-residues). Alginate lyases define a class of enzymes that cleave the glycosidic bond of alginate by ß-elimination. They are classified according to their ability to recognize the distribution of M- and G-residues and are named M-, G- or MG-lyases. In the CAZy database, alginate lyases have been grouped by sequence similarity into seven distinct polysaccharide lyase families. The polysaccharide lyase family PL6 is subdivided into three subfamilies. Subfamily PL6_1 includes three biochemically characterized enzymes (two alginate lyases and one dermatan sulfatase lyase). No characterized enzymes have been described in the two other subfamilies (PL6_2 and PL6_3). To improve the prediction of polysaccharide-lyase activity in the PL6 family, we re-examined the classification of the PL6 family and biochemically characterized a set of enzymes reflecting the diversity of the protein sequences. Our results show that subfamily PL6_1 includes two dermatan sulfates lyases and several alginate lyases that have various substrate specificities and modes of action. In contrast, subfamilies PL6_2 and PL6_3 were found to contain only endo-poly-MG-lyases.

Comparative characterization of two GDP-mannose dehydrogenase genes from Saccharina japonica (Laminariales, Phaeophyceae).

BACKGROUND: Saccharina japonica is an important commercial brown seaweed, its main product is alginate, which is used in food, textile and by the cosmetic and pharmaceutical industries. GDP-mannose dehydrogenase (GMD) is the key enzyme involved in the synthesis of alginate. However, little is known about GMD in S. japonica. Here we report comparative biochemical analysis of two GMD genes in S. japonica. RESULTS: Two GMD genes from S. japonica (Sjgmd1, Sjgmd2) were cloned. The open reading frame lengths of Sjgmd1, Sjgmd2 are 963 bp and 948 bp, respectively. Alignment analysis showed that the two SjGMD sequences shared 79.38 % identity. Both proteins possess the GGxCLPKDV and GxGxVG sequence motifs characteristic of the short-chain dehydrogenase/reductase superfamily. The optimum temperatures for SjGMDs were 30 °C (SjGMD1) and 20 °C (SjGMD2), and the optimum pH values were 8.0 (SjGMD1) and 8.25 (SjGMD2). Kinetic analysis demonstrated the Km values for the substrate GDP-mannose were 289 µM (SjGMD1) and 177 µM (SjGMD2), and the Km values for the cofactor NAD(+) were 139 µM (SjGMD1) and 195 µM (SjGMD2). The metal iron Zn(2+) is a potent inhibitor of SjGMD1 and SjGMD2. Real-time PCR analysis showed that heat and desiccation treatments resulted in a significant increase in Sjgmd1 and Sjgmd2 transcript abundance, suggesting that the SjGMDs are directly involved in the acclimitisation of S. japonica to abiotic stresses. CONCLUSION: Our work identified two novel genes encoding GMD in S. japonica, comparatively characterized their structural characteristics and enzyme kinetics, and revealed the function of GMD in the stress adaptability of S. japonica. The knowledge obtained here enriched our understanding of the alginate synthesis mechanism in S. japonica, and may promote further research on functional differences between GMD genes.

The cell-wall active mannuronan C5-epimerases in the model brown alga Ectocarpus: From gene context to recombinant protein.

Mannuronan C5-epimerases (ManC5-Es) catalyze in brown algae the remodeling of alginate, a major cell-wall component which is involved in many biological functions in these organisms. ManC5-Es are present as large multigenic families in brown algae, likely indicating functional specificities and specializations. ManC5-Es control the distribution pattern of (1-4) linked ß-d-mannuronic acid (M) and α-l-guluronic acid (G) residues in alginates, giving rise to widely different polysaccharide compositions and sequences, depending on tissue, season, age, or algal species. As such they are also a source of powerful new tools for the biotechnological and enzymatic processing of alginates, to match the growing interest for food hydrocolloids and in biomedical and nanotechnological applications. We report here the first heterologous production of a ManC5-E of brown algal origin that is successfully refolded in an active form. The activity was measured by 1H NMR and by an indirect enzymatic assay using a known bacterial alginate lyase. The transcript expression as a function of the developmental program of the brown alga Ectocarpus, together with the bioinformatic analyses of the corresponding gene context of this multigenic family, is also presented.

Molecular and biochemical characterization of mannitol-1-phosphate dehydrogenase from the model brown alga Ectocarpus sp.

The sugar alcohol mannitol is important in the food, pharmaceutical, medical and chemical industries. It is one of the most commonly occurring polyols in nature, with the exception of Archaea and animals. It has a range of physiological roles, including as carbon storage, compatible solute, and osmolyte. Mannitol is present in large amounts in brown algae, where its synthesis involved two steps: a mannitol-1-phosphate dehydrogenase (M1PDH) catalyzes a reversible reaction between fructose-6-phosphate (F6P) and mannitol-1-phosphate (M1P) (EC 1.1.1.17), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 3.1.3.22). Analysis of the model brown alga Ectocarpus sp. genome provided three candidate genes for M1PDH activities. We report here the sequence analysis of Ectocarpus M1PDHs (EsM1PDHs), and the biochemical characterization of the recombinant catalytic domain of EsM1PDH1 (EsM1PDH1cat). Ectocarpus M1PDHs are representatives of a new type of modular M1PDHs among the polyol-specific long-chain dehydrogenases/reductases (PSLDRs). The N-terminal domain of EsM1PDH1 was not necessary for enzymatic activity. Determination of kinetic parameters indicated that EsM1PDH1cat displayed higher catalytic efficiency for F6P reduction compared to M1P oxidation. Both activities were influenced by NaCl concentration and inhibited by the thioreactive compound pHMB. These observations were completed by measurement of endogenous M1PDH activity and of EsM1PDH gene expression during one diurnal cycle. No significant changes in enzyme activity were monitored between day and night, although transcription of two out of three genes was altered, suggesting different levels of regulation for this key metabolic pathway in brown algal physiology.

Cooperativity and evolution of Tetrahymena two-domain arginine kinase.

Tetrahymena pyriformis contains two arginine kinases, a 40-kDa enzyme (AK1) with a myristoylation signal sequence at the N-terminus and a two-domain 80-kDa enzyme (AK2). The former is localized mainly in cilia and the latter is in the cytoplasm. AK1 was successfully synthesized using an insect cell-free protein synthesis system and subjected to peptide mass fingerprinting (PMF) analysis. The masses corresponding to unmodified N-terminal tryptic peptide or N-terminal myristoylated peptide were not observed, suggesting that N-terminal peptides were not ionized in this analysis. We performed PMF analyses for two other phosphagen kinases (PKs) with myristoylation signals, an AK from Nematostella vectensis and a PK from Ectocarpus siliculosus. In both cases, the myristoylated, N-terminal peptides were clearly identified. The differences between the experimental and theoretical masses were within 0.0165-0.0583 Da, supporting the accuracy of the identification. Domains 1 and 2 of Tetrahymena two-domain AK2 were expressed separately in Escherichia coli and the extent of cooperativity was estimated on the basis of their kinetic constants. The results suggested that each of the domains functions independently, namely no cooperativity is displayed between the two domains. This is in sharp contrast to the two-domain AK from Anthopleura.

Mannitol metabolism in brown algae involves a new phosphatase family.

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase and a mannitol-1-phosphatase (M1Pase). Genome analysis of the brown algal model Ectocarpus siliculosus allowed identification of genes potentially involved in the mannitol cycle. Among these, two genes coding for haloacid dehalogenase (HAD)-like enzymes were suggested to correspond to M1Pase activity, and thus were named EsM1Pase1 and EsM1Pase2, respectively. To test this hypothesis, both genes were expressed in Escherichia coli. Recombinant EsM1Pase2 was shown to hydrolyse the phosphate group from mannitol-1-phosphate to produce mannitol but was not active on the hexose monophosphates tested. Gene expression analysis showed that transcription of both E. siliculosus genes was under the influence of the diurnal cycle. Sequence analysis and three-dimensional homology modelling indicated that EsM1Pases, and their orthologues in Prasinophytes, should be seen as founding members of a new family of phosphatase with original substrate specificity within the HAD superfamily of proteins. This is the first report describing the characterization of a gene encoding M1Pase activity in photosynthetic organisms.