Serine Hydroxyl Research Articles

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Silk sericin as a bio-initiator for grafting from synthesis of polylactide via ring-opening polymerization

In this study, a new graft copolymer (SS-g-PLA) was constructed by combining sericin and polylactide (PLA) via ring-opening polymerization (ROP) having Sn(Oct)2 as a catalyst. In this synthetic strategy based on the grafting from concept, sericin molecule acts as a bio-initiator where its hydroxyl groups existing in serine units. The effects of polymerization parameters, including reaction temperature, the concentration of catalyst and sericin, on the copolymer structure and properties were investigated. XPS spectra of SS-g-PLA copolymer showed C1S and N1S signals clearly and differed form that of the neat PLA. 2D HMBC NMR spectra showed correlation assigned to the carbon atom of PLA polymer chains and hydrogen atom in hydroxyl group of serine. Molecular weight of the graft copolymer by GPC was in range of 4000–5600 g mol−1. From soxhlet extraction, the results showed that PLA content in the graft copolymer molecules increased with increasing sericin content. The weight ratio of PLA to sericin in the SS-g-PLA5, SS-g-PLA10 and SS-g-PLA15 graft copolymers were 0.6:1, 1.4:1 and 2.1:1, respectively. By nitrogen analyzer,%protein increased significantly with increasing sericin content. Thermal analysis showed that the graft copolymer presented a single glass-transition temperature (Tg) belonging to PLA. This Tg decreased with increasing sericin content. The thermal degradation temperature (Td) of SS-g-PLA copolymer decreased to about 60 °C. Furthermore, SS-g-PLA is expected to utilize in biomedical applications.

Switching on a Nontraditional Enzymatic Base—Deprotonation by Serine in the ent-Kaurene Synthase from Bradyrhizobium japonicum

Terpene synthases often catalyze complex carbocation cascade reactions. It has been previously shown that single residue switches involving replacement of a key aliphatic residue with serine or threonine can "short-circuit" such reactions, presumed to act indirectly via dipole stabilization of intermediate carbocations. Here a similar switch was found in the structurally characterized ent-kaurene synthase from Bradyrhizobium japonicum. Application of a recently developed computational approach to terpene synthases, TerDockin, surprisingly indicates direct action of the introduced serine hydroxyl as a catalytic base. Notably, this model suggests alternative interpretation of previous results, and potential routes towards reengineering terpene synthase activity more generally.

Synthesis and biological roles of O-glycans in insects.

Protein O-glycosylation is the attachment of carbohydrate structures to the oxygen atom in the hydroxyl group of Serine and Threonine residues. This post-translational modification is commonly found on the majority of proteins trafficking through the secretory pathway and is reported to influence protein characteristics such as folding, secretion, stability, solubility, oligomerization and intracellular localization. In addition, O-glycosylation is essential for cell-cell interactions, protein-protein interactions and many biological processes, such as stress response, immunization, phosphorylation, ubiquitination, cell division, metabolism and cell signaling. The availability of sequenced genomes and genetic tools to create mutants with clear phenotypes makes insects an interesting model system to study O-glycosylation. In this review, we provide an overview of the current knowledge of O-glycosylation, mainly obtained from the model organism Drosophila melanogaster, with a focus on the synthesis and biological roles of the common O-glycans in insects.

Congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) is a genetically heterogeneous and clinically polymorphic group of diseases caused by defects in various enzymes, the synthesis and processing of N-linked glycans or oligosaccharides into glycoproteins. Approximately half of all proteins expressed in cells are glycosylated to achieve their full functionality. Basically there are 2 variants of glycosylation: N-glycosylation and O-glycosylation. N-glycans are bound to the amide group of aspartine, whereas O-glycans are bonded to the hydroxyl group of serine or threonine. Synthesis of N-glycans occurs in 3 stages: the formation of nucleotide-linked sugars, assembly (in the cytosol and endoplasmic reticulum) and treatment (in the Golgi apparatus). Synthesis of O-glycans occurs mainly in the Golgi apparatus. The most frequently identified types of CDG are associated with a defect in the N-glycosylation pathway. CDGs are typically multisystem disorders with varying clinical manifestations such as hepatomegaly, cholestasis, liver failure, developmental delay, hypotonia, convulsions, facial dysmorphism and gastrointestinal disorders. Also histological findings showed liver fibrosis, malformation of the ducts, cirrhosis, and steatosis. CDGs typically present in the first months of life, and about 20% of patients do not survive to 5 years. The first line of CDG screening is based on the analysis of N-glycosylation of transf ferin. Exome sequencing or targeted gene panel is used for diagnosis. Several CDG subtypes are amenable to teraphy with mannose and galactose.

Involvement of O-GlcNAcylation in the Skeletal Muscle Physiology and Physiopathology: Focus on Muscle Metabolism

Skeletal muscle represents around 40% of whole body mass. The principal function of skeletal muscle is the conversion of chemical energy toward mechanic energy to ensure the development of force, provide movement and locomotion, and maintain posture. This crucial energy dependence is maintained by the faculty of the skeletal muscle for being a central place as a “reservoir” of amino acids and carbohydrates in the whole body. A fundamental post-translational modification, named O-GlcNAcylation, depends, inter alia, on these nutrients; it consists to the transfer or the removal of a unique monosaccharide (N-acetyl-D-glucosamine) to a serine or threonine hydroxyl group of nucleocytoplasmic and mitochondrial proteins in a dynamic process by the O-GlcNAc Transferase (OGT) and the O-GlcNAcase (OGA), respectively. O-GlcNAcylation has been shown to be strongly involved in crucial intracellular mechanisms through the modulation of signaling pathways, gene expression, or cytoskeletal functions in various organs and tissues, such as the brain, liver, kidney or pancreas, and linked to the etiology of associated diseases. In recent years, several studies were also focused on the role of O-GlcNAcylation in the physiology and the physiopathology of skeletal muscle. These studies were mostly interested in O-GlcNAcylation during muscle exercise or muscle-wasting conditions. Major findings pointed out a different “O-GlcNAc signature” depending on muscle type metabolism at resting, wasting and exercise conditions, as well as depending on acute or long-term exhausting exercise protocol. First insights showed some differential OGT/OGA expression and/or activity associated with some differential stress cellular responses through Reactive Oxygen Species and/or Heat-Shock Proteins. Robust data displayed that these O-GlcNAc changes could lead to (i) a differential modulation of the carbohydrates metabolism, since the majority of enzymes are known to be O-GlcNAcylated, and to (ii) a differential modulation of the protein synthesis/degradation balance since O-GlcNAcylation regulates some key signaling pathways such as Akt/GSK3β, Akt/mTOR, Myogenin/Atrogin-1, Myogenin/Mef2D, Mrf4 and PGC-1α in the skeletal muscle. Finally, such involvement of O-GlcNAcylation in some metabolic processes of the skeletal muscle might be linked to some associated diseases such as type 2 diabetes or neuromuscular diseases showing a critical increase of the global O-GlcNAcylation level.

GENETIC POLYMORPHISMS INFLUENCING EFFICACY AND SAFETY OF METHOTREXATE IN RHEUMATOID ARTHRITIS

Objective: This review will summarize pharmacogenetic studies of single nucleotide polymorphisms in genes coding enzymes of methotrexate (MTX) pathway, related to its response and toxicity in rheumatoid arthritis (RA). In addition, this review focuses on the racial and ethnic differences in distribution of the polymorphisms in genes related to efficacy and toxicity of MTX in RA.Methods: Articles were searched using Pubmed database using the search term “pharmacogenetics and MTX and arthritis.” The search revealed 72 articles, of which 27 were given special importance, due to open-access.Results: Many genes and single nucleotide polymorphisms are investigated in this context, and the highlighting genes are ATP-binding cassette proteins (ABCB1) reduced folate carrier (RFC), methylenetetrahydrofolatereductase (MTHFR), gamma-glutamyl hydrolase (GGH), serine hydroxyl methyltransferase (SHMT), 5-aminoimidazole-4-carboxamide ribonucleotidetransformylase (ATIC), methionine synthase reductase (MTRR), methionine synthase (MS), adenosine monophosphate deaminase1 (AMPD1), inosine triphosphate pyrophosphatase (ITPA). The study highlighted RFC-1 80AA, ITPA 94CC, and AMPD1 347CC as responders. The allelic types prone to toxicity were MTHFR 677TT, MTRR 2756AA, MS 66GG, SHMT 1420CC, ATIC 347GG, and thymidylate synthase *3/*2. The genotypes reported as non-responders were ABCB1 3434TT, MTHFR 1298AA, DHFR A317G, GGH 16CC, and GGH 401TT.Conclusion: Although these studies highlight inconsistency in results, due to the difference in sample size and assessment parameters and racial and ethnic differences, larger prospective studies are essential to reach the cornerstone of the concept of personalized medicine.

Neuronal O-GlcNAc transferase regulates appetite, body weight, and peripheral insulin resistance.

The ogt gene encodes O-linked N-acetylglucosamine transferase (O-GlcNAc transferase [OGT]) that catalyzes the transfer of β-N-acetylglucosamine (GlcNAc) from the uridine-diphosphate-GlcNAc to the hydroxyl group of serine or threonine residues of nucleocytoplasmic proteins. This process is a common protein posttranslational modification, called protein O-GlcNAcylation, which is a known intracellular sensor of glucose metabolism and plays an important role in regulating cellular signaling, transcription, and metabolism. However, little is known about the function of OGT in the brain. Here, we report that the CaMKIIα promoter-dependent neuronal knockout (KO) of OGT in adult mice led to short-term overeating, body weight gain, and peripheral insulin resistance. These phenotype changes were accompanied by marked elevation of serum insulin and leptin levels and neuronal cell death, including the loss of leptin receptor-expressing neurons, in the hypothalamus. The neuronal OGT KO exacerbated obesity and insulin resistance induced by high-fat diet. Surprisingly, the peripheral insulin resistance induced by neuronal OGT KO was reversed at its own 2-3months after OGT KO, and the mice even showed increased insulin sensitivity several months later. These findings reveal an important role of neuronal OGT in the regulation of feeding behavior, body weight, and peripheral insulin sensitivity.

Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

Rational design of carbamate-based dual binding site and central AChE inhibitors by a “biooxidisable” prodrug approach: Synthesis, in vitro evaluation and docking studies

Herein, we report a new class of dual binding site AChE inhibitor 4 designed to exert a central cholinergic activation thanks to a redox-activation step of a prodrug precursor 3. Starting from potent pseudo-irreversible quinolinium salts AChE inhibitors 2 previously reported, a new set of diversely substituted quinolinium salts 2a-p was prepared and assayed for their inhibitory activity against AChE. Structure-activity relationship (SAR) analysis of 2a-p coupled with molecular docking studies allowed us to determine which position of the quinolinium scaffold may be considered to anchor the phtalimide fragment presumed to interact with the peripheral anionic site (PAS). In addition, molecular docking provided insight on the linker length required to connect both quinolinium and phatlimide moieties without disrupting the crucial role of quinolinium salt moiety within the catalytic active site (CAS); namely placing the carbamate in the correct position to trigger carbamylation of the active-site serine hydroxyl. Based on this rational design, the putative dual binding site inhibitor 4 and its prodrug 3 were synthesized and subsequently evaluated in vitro against AChE. Pleasingly, whereas compound 4 showed to be a highly potent inhibitor of AChE (IC50 = 6 nM) and binds to AChE-PAS to the same extent as donepezil, its prodrug 3 revealed to be inactive (IC50 > 10 μM). These preliminary results constitute one of the few examples of carbamate-based dual binding site AChE inhibitors.

The Effect of Single and Multiple SERAT Mutants on Serine and Sulfur Metabolism.

The gene family of serine acetyltransferases (SERATs) constitutes an interface between the plant pathways of serine and sulfur metabolism. SERATs provide the activated precursor, O-acetylserine for the fixation of reduced sulfur into cysteine by exchanging the serine hydroxyl moiety by a sulfhydryl moiety, and subsequently all organic compounds containing reduced sulfur moieties. We investigate here, how manipulation of the SERAT interface results in metabolic alterations upstream or downstream of this boundary and the extent to which the five SERAT isoforms exert an effect on the coupled system, respectively. Serine is synthesized through three distinct pathways while cysteine biosynthesis is distributed over the three compartments cytosol, mitochondria, and plastids. As the respective mutants are viable, all necessary metabolites can obviously cross various membrane systems to compensate what would otherwise constitute a lethal failure in cysteine biosynthesis. Furthermore, given that cysteine serves as precursor for multiple pathways, cysteine biosynthesis is highly regulated at both, the enzyme and the expression level. In this study, metabolite profiles of a mutant series of the SERAT gene family displayed that levels of the downstream metabolites in sulfur metabolism were affected in correlation with the reduction levels of SERAT activities and the growth phenotypes, while levels of the upstream metabolites in serine metabolism were unchanged in the serat mutants compared to wild-type plants. These results suggest that despite of the fact that the two metabolic pathways are directly connected, there seems to be no causal link in metabolic alterations. This might be caused by the difference of their pool sizes or the tight regulation by homeostatic mechanisms that control the metabolite concentration in plant cells. Additionally, growth conditions exerted an influence on metabolic compositions.

Simplified beta-glycosylation of peptides

A simple and effective activating system for S-phenyl thioglycosides, namely N-iodosuccinimide and catalytic copper(I) triflate, promotes beta-O-glycosylation at the serine and threonine hydroxyls of “mono-,” di-, and tripeptides. The same activator combination promotes carboxamide beta-N-glycosylation of asparagine-containing mono-, di, and tri-peptides, as well as a nucleoside carboxamide and a sulfonamide. An important feature of the copper(I) triflate method is that undesired amide O-glycosylation is largely circumvented. For both sets of biologically important acceptor sites (HO– and –CONH2), a beta-GlcNAc-equivalent donor is demonstrated to provide the linkages efficiently. Streamlined deprotection sequences have been developed based on global hydrogenolysis that lead smoothly to the parent glycopeptides. The core glycopeptide region for biological protein N-glycosylation, represented by N4-(β-N-acetyl-D-2-glucosaminyl)-Asp-Gly-Thr-OH, has been prepared in solution, purified, and characterized as the fully deprotected (mono)glycosylated tripeptide.

Synthesis and characterization of cross linked enzyme aggregates of serine hydroxyl methyltransferase from Idiomerina leihiensis

A thermo-stable purified serine hydroxymethyltransferase (SHMT; 418 AA) was used for the carrier free immobilization using pectin as a coach molecule and formaldehyde as a cross-linker. The purified protein was cross linked with formaldehyde in the presence of pectin to form stable and active aggregates. The cross-linked enzyme aggregates [CLEAs] of SHMT showed improved catalytic properties and reusability. The SHMT-CLEAs showed a noteworthy change in the thermo-stability and activity compared to its free counterpart. The optimum activity for free SHMT was reported at 55 °C and pH 7.5 which SHMT CLEAs showed maximum activity at 60 °C and pH 8.0. Similarly, the CLEAs were noticed to increase the thermo-stability in comparison to free enzyme. The divalent salt ion Ca2+ and Ba2+ were found to enhance the activity at 1 and 5 mM of concentrations while Ni+, Co2+ and Zn2+ strongly inhibited the activity of both free as well as CLEAs. The Vmax and km values for free SHMT were recorded to be 1.21 μM s−1 and 272 μM while for CLEAs Vmax 1.42 μM s−1 and km 248.6 μM was recorded. Thus, a 120% increase in the Vmax was recorded for SHMT-CLEAs. The CLEAs were also found to be more stable at pH 6.5 and 8.5 pHs and retained 50% of its original activity for 180 and 200 min respectively. The CLEAs also retained 72% of its activity after 12 repetitive cycles of d-phenylserine hydrolysis. Also, the synthesized CLEAs retained more than 60% of its original activity after 10 days of incubation at 25 °C in comparison to free enzyme which loses more than 90% of its residual activity. Thus, with improved thermostability and activity the CLEAs of SHMT can be used repetitively at industrial scale for the synthesis of commercially important amino acids.

The Molecular Mechanism of Intermolecular Signal Transduction in Cystathionine‐β‐Synthase (CBS)

Human cystathionine‐β‐synthase (hCBS) is a pyridoxal phosphate‐dependent (PLP) enzyme that contains heme and catalyzes the β‐replacement reactions of serine (L‐Ser) by homocysteine (L‐Hcy) to produce cystathionine. Mutation in this key mammalian enzyme causes hyperhomocysteinemia, which leads to aggressive arterial diseases, a clinical phenotype observed in patients. Although the role of PLP is similar to other PLP dependent enzymes that catalyze the exchange of the hydroxyl group of serine to thiolate in homocysteine, little is known about the role that heme plays in regulating the catalytic process. My project seeks to develop a continuous, kinetic assay to measure CBS activity. Two approaches are being explored: 1) A coupled kinetic assay, in which the cystathionine produced by CBS is used by Cystathionine‐β‐lyase (hCBL) to generate pyruvate, and pyruvate production is measured phototrically; 2) A novel nuclear magnetic resonance assay, in which we will monitor the direct production of cystathionine over time. These methods will be compared to the standard, but discontinuous, radiometric assay. These assays will be used to understand the effect of small ligand binding to the heme and point mutations at the heme cofactor in relation to the enzyme activity. The long‐term goal of this work is to use time‐resolved absorption and magnetic circular dichroism spectroscopy to understand how small ligand binding to the heme induces conformational changes at the PLP active site in CBS and to measure activity of CBS with various mutated active site residues.Support or Funding InformationArnold and Mabel Beckman Foundation; NIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Dynamics Study of the Complex Formation of TEM-Type β-Lactamases with Substrates and Inhibitors

The complex formation of TEM-1 β-lactamase and its three mutant forms TEM-32, TEM-37, and TEM-39 with substrates cephalothin and CENTA and serine beta-lactamase inhibitors sulbactam, tazobactam, and clavulanic acid is studied using the methods of molecular dynamics. It is found that the stability of the complexes is caused by the electrostatic attraction between the deprotonated carboxyl group of the β-lactam ring of the substrate (inhibitor) and the positively charged amino groups of the lysine 234 and 73 residues, located in the active site of the enzymes. The formation of a hydrogen bond between this substrate group or its carbonyl oxygen with the hydroxyl group of the catalytic serine 70 residue and also between the negatively charged substituent groups and the positive charge region formed by the arginine 244 guanidine group and the asparagine 276 amino group is observed for some complexes. The binding energy of CENTA with TEM-1 β-lactamase is below the analogous binding energy of cephalothin, which is confirmed by the values of the Michaelis constants, determined experimentally. It is also found that the inhibitors bind to the mutant forms of β-lactamases related to the inhibitor-resistant phenotype, with higher affinity than TEM-1 β-lactamase.

Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity.

Protein glycosylation is a ubiquitous post-translational modification that plays crucial roles in modulating biological recognition events in development and physiology. Human O-GlcNAc transferase (OGT) is an intracellular enzyme responsible for O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, and the deregulation of OGT activity occurs in cancer, diabetes, and neurodegenerative disease. Here we develop a single quantum dot (QD)-based nanosensor for sensitive OGT assay. We design a Cy5/biotin-modified peptide with a serine hydroxyl group for sensing OGT and a protease site adjacent to the glycosylation site for proteinase cleavage, with a universal nonradioactive UDP-GlcNAc as the sugar donor and a Cy5/biotin-modified peptide as the substrate. In the presence of OGT, it catalyzes the glycosylation reaction to generate a glycosylated peptide that is a protease-protection peptide. The resultant glycosylated Cy5/biotin-modified peptides may assemble on the surface of the streptavidin-coated QD to obtain a QD-peptide-Cy5 nanostructure in which the fluorescence resonance energy transfer (FRET) from the QD to Cy5 can occur, leading to the emission of Cy5 which can be quantified by single-molecule detection. This method exhibits high sensitivity with a limit of detection of 3.47 × 10-13 M, and it is very simple and straightforward without the involvement of any enzyme purification, radioisotope-labeled sugar donors, specific antibodies, and the synthesis of fluorescent UDP-GlcNAc analogues. Moreover, this method can be used for enzyme kinetic analysis, quantitative detection of cellular OGT activity, and the screening of OGT inhibitors, holding great potential for further application in drug discovery and clinical diagnosis.

Targeted Mass Spectrometry Approach Enabled Discovery of O-Glycosylated Insulin and Related Signaling Peptides in Mouse and Human Pancreatic Islets.

O-Linked glycosylation often involves the covalent attachment of sugar moieties to the hydroxyl group of serine or threonine on proteins/peptides. Despite growing interest in glycoproteins, little attention has been directed to glycosylated signaling peptides, largely due to lack of enabling analytical tools. Here we explore the occurrence of naturally O-linked glycosylation on the signaling peptides extracted from mouse and human pancreatic islets using mass spectrometry (MS). A novel targeted MS-based method is developed to increase the likelihood of capturing these modified signaling peptides and to provide improved sequence coverage and accurate glycosite localization, enabling the first large-scale discovery of O-glycosylation on signaling peptides. Several glycosylated signaling peptides with multiple glycoforms are identified, including the first report of glycosylated insulin-B chain and insulin-C peptide and BigLEN. This discovery may reveal potential novel functions as glycosylation could influence their conformation and biostability. Given the importance of insulin and its related peptide hormones and previous studies of glycosylated insulin analogues, this natural glycosylation may provide important insights into diabetes research and therapeutic treatments.

Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide microarray

O-GlcNAcylation is a post-translational modification resulting from the addition of an N-acetylglucosamine moiety to the hydroxyl groups of serine and threonine residues of nuclear and cytoplasmic proteins. In addition, O-GlcNAcylated proteins can be phosphorylated, which suggests the possibility for crosstalk between O-GlcNAcylation and phosphorylation. Dysregulation of O-GlcNAcylation affects cell signaling, transcriptional regulation, cell cycle control and can e.g. lead to tumorigenesis and tumor metastasis. There is a strong demand for efficient analytical techniques to better detect and investigate this abundant modification and its role in cancer. Herein we demonstrated the utility of an O-GlcNAcylated peptide array to examine O-GlcNAcase (OGA) activity and substrate specificity of both purified protein as well cell lysates of different cancer cell lines. Using this microarray, we clearly observed OGA activity and also inhibition thereof by OGA inhibitor thiamet G. Interestingly, different levels of OGA activity were observed of lysates derived from different cancer cell lines. This suggests that the tool may be useful in cancer research and biomarker development.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos.

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus.

Biodegradation of phthalic acid esters (PAEs) and in silico structural characterization of mono-2-ethylhexyl phthalate (MEHP) hydrolase on the basis of close structural homolog

Three bacterial strains capable of degrading phthalates namely Pseudomonas sp. PKDM2, Pseudomonas sp. PKDE1 and Pseudomonas sp. PKDE2 were isolated and characterized for their degradative potential. These strains efficiently degraded 77.4%–84.4% of DMP, 75.0%–75.7% of DEP and 71.7%–74.7% of DEHP, initial amount of each phthalate is 500mgL−1 of each phthalate, after 44h of incubation. GC–MS results reveal the tentative DEHP degradation pathway, where hydrolases mediate the breakdown of DEHP to phthalic acid (PA) via an intermediate MEHP. MEHP hydrolase is a serine hydrolase which is involved in the reduction of the MEHP to PA. The predicted 3D model of MEHP hydrolase from Pseudomonas mosselii was docked with phthalate monoesters (PMEs) such as MEHP, mono-n-hexyl phthalate (MHP), mono-n-butyl phthalate (MBP) and mono-n-ethyl phthalate (MEP), respectively. Docking results show the distance between the carbonyl carbon of respective phthalate monoester and the hydroxyl group of catalytic serine lies in the range of 2.9 to 3.3Å, which is similar to the ES complex of other serine hydrolases. This structural study highlights the interaction and the role of catalytic residues of MEHP hydrolase involved in the biodegradation of PMEs to phthalate.