Structural Characterization of L-Galactose Dehydrogenase: An Essential Enzyme for Vitamin C Biosynthesis.

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (ß/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata.

α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-l-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-l-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

First characterization of fucosidases in spiders.

l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 µM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

High throughput techniques to reveal the molecular physiology and evolution of digestion in spiders.

BACKGROUND: Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput ("-omics") techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). RESULTS: We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. CONCLUSION: All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands.

Biochemical, transcriptomic and proteomic analyses of digestion in the scorpion Tityus serrulatus: insights into function and evolution of digestion in an ancient arthropod.

Scorpions are among the oldest terrestrial arthropods and they have passed through small morphological changes during their evolutionary history on land. They are efficient predators capable of capturing and consuming large preys and due to envenomation these animals can become a human health challenge. Understanding the physiology of scorpions can not only lead to evolutionary insights but also is a crucial step in the development of control strategies. However, the digestive process in scorpions has been scarcely studied. In this work, we describe the combinatory use of next generation sequencing, proteomic analysis and biochemical assays in order to investigate the digestive process in the yellow scorpion Tityus serrulatus, mainly focusing in the initial protein digestion. The transcriptome generated database allowed the quantitative identification by mass spectrometry of different enzymes and proteins involved in digestion. All the results suggested that cysteine cathepsins play an important role in protein digestion. Two digestive cysteine cathepsins were isolated and characterized presenting acidic characteristics (pH optima and stability), zymogen conversion to the mature form after acidic activation and a cross-class inhibition by pepstatin. A more elucidative picture of the molecular mechanism of digestion in a scorpion was proposed based on our results from Tityus serrulatus. The midgut and midgut glands (MMG) are composed by secretory and digestive cells. In fasting animals, the secretory granules are ready for the next predation event, containing enzymes needed for alkaline extra-oral digestion which will compose the digestive fluid, such as trypsins, astacins and chitinase. The digestive vacuoles are filled with an acidic proteolytic cocktail to the intracellular digestion composed by cathepsins L, B, F, D and legumain. Other proteins as lipases, carbohydrases, ctenitoxins and a chitolectin with a perithrophin domain were also detected. Evolutionarily, a large gene duplication of cathepsin L occurred in Arachnida with the sequences from ticks being completely divergent from other arachnids probably due to the particular selective pressures over this group.

Cysteine cathepsins as digestive enzymes in the spider Nephilengys cruentata.

Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles.

Characterization of α-L-fucosidase and other digestive hydrolases from Biomphalaria glabrata.

Schistosoma mansoni is one of the major agents of the disease Schistosomiasis, which is one of the major global public health concerns. Biomphalaria glabrata is an obligate intermediate mollusc host of S. mansoni. Although the development of S. mansoni occurs in the snail hepatopancreas, studies that focus on this organ remain limited. In this study, we biochemically identified five distinct carbohydrases (amylase, maltase, α-glucosidase, trehalase, and α-L-fucosidase), lipases, and peptidases in the B. glabrata hepatopancreas and focused on the isolation and characterization of the activity of α-L-fucosidase. The isolated α-L-fucosidase has a molecular mass of 141 kDa, an optimum pH of 5.8, and is inhibited by Tris, fucose, and 1-deoxyfuconojirimycin. B. glabrata α-L-fucosidase is an exoglycosidase that can hydrolyze the natural substrate fucoidan to fucose residues. It presented Km values of 48.4 µM to 4-Methylumbelliferyl α-L-fucopyranoside and 0.55 mM to p-nitrophenyl-α-L-fucopyranoside. Thus, α-L-fucosidase has a high activity in the hepatopancreas of B. glabrata, and the differential expression of this enzyme between susceptible and resistant strains indicates that besides its digestive role, α-L-fucosidase may also be important in host/parasite interactions.

Transcriptome sequencing and developmental regulation of gene expression in Anopheles aquasalis.

BACKGROUND: Anopheles aquasalis is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to Plasmodium vivax and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of An. aquasalis genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the An. aquasalis transcriptome. METHODOLOGY/PRINCIPAL FINDINGS: A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥ 2 sequences, and 1999 singletons. The majority of the An. aquasalis transcripts encode proteins with their closest counterparts in another neotropical malaria vector, An. darlingi. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced An. aquasalis proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed. CONCLUSIONS/SIGNIFICANCE: This study represents the first large-scale effort to sequence the transcriptome of An. aquasalis. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The An. aquasalis transcriptome is accessible at http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx.

Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants.

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Four insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera, and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization using different colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe p-nitroanilide). Chloromethyl ketones (TPCK, N- alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N- carbobenzoxy-Gly-Gly-Phe-CK) inactivated all chymotrypsins tested. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1) s(-1); Z-GGF-CK, 150 to 450 M(-1) s(-1)) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1) s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa value. This is proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed to be an adaptation to the presence of dietary ketones.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.