Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.     

The unusual di‐domain structure of Dunaliella salina glycerol‐3‐phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di‐domain glycerol‐3‐phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase‐like domain fused to the canonical glycerol‐3‐phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co‐factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.     

Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

Characterization of a thermostable glucose dehydrogenase with strict substrate specificity from a hyperthermophilic archaeon Thermoproteus sp. GDH-1

A hyperthermophilic archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity. The enzyme (TgGDH) was purified and found to display a strict preference for d-glucose. The gene was cloned and expressed in Escherichia coli, resulting in the production of a soluble and active protein. Recombinant TgGDH displayed extremely high thermostability and an optimal temperature higher than 85 °C, in addition to its strict specificity for d-glucose. Despite its thermophilic nature, TgGDH still exhibited activity at 25 °C. We confirmed that the enzyme could be applied for glucose measurements at ambient temperatures, suggesting a potential of the enzyme for use in measurements in blood samples.     

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.     

Characterization of malate dehydrogenase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrobaculum islandicum</Emphasis>

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K _m values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80°C and pH 8.0) were 15 and 86 μM, respectively, with specific activity as high as 470 U mg⁻¹. Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD⁺ were used as substrates and was not detected with NADP⁺. A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.     

The synthesis of N‐benzoylindoles as inhibitors of rat erythrocyte glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase

Glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) play an important function in various biochemical processes as they generate reducing power of the cell. Thus, metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combinational therapeutic approaches. In this study, N‐benzoylindoles 9a‐ ‐ 9d , which form the main framework of many natural indole derivatives such as indomethacin and N‐benzoylindoylbarbituric acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of G6PD and 6PGD. The N‐benzoylindoles inhibited the enzymatic activity with IC₅₀ in the range of 3.391505 μM for G6PD and 2.19–990 μM for 6PGD.     

Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Short‐chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2‐keto‐3‐deoxy‐d ‐gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG‐producing SDR enzymes (NADPH‐dependent A1‐R and NADH‐dependent A1‐R′) involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes—KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)—prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5‐diketo‐3‐deoxy‐d ‐gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X‐ray crystallography. Both PcaKduD and SpyDhuD consist of a three‐layered structure, α/β/α, with a coenzyme‐binding site in the Rossmann fold; similar to enzymes A1‐R and A1‐R′, both arrange the two short and long loops close to the coenzyme‐binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1‐R′ but not A1‐R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2′ coenzyme region were determined to be structurally crucial for dual‐coenzyme specificity in PcaKduD by structural comparison of the NADH‐ and NADPH‐specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual‐coenzyme specificity. Proteins 2016; 84:934–947. © 2016 Wiley Periodicals, Inc.     

Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Glucose‐6‐phosphate dehydrogenase encapsulated in silica‐based hydrogels for operation in a microreactor

The future of hydrogen as fuel strongly depends on the possibility to produce it in an economic and clean way. Hydrogen can be produced from carbohydrates and water under mild conditions by means of a multistep synthetic pathway (13 enzymes) with very high yield. Crossover inhibitions and different optimal conditions of involved enzymes hinder the use of one‐pot approach. Immobilization of enzymes in coupled individual reactors may avoid this problem. This work deals with the immobilization in silica‐based hydrogels of one key enzyme of this pathway: glucose 6‐phosphate dehydrogenase from Leuconostoc mesenteroides. The carriers were prepared with an ethylene glycol‐modified silane, two polymers (polyethylene oxide and Pluronic®) and amino groups created by 3‐aminopropyltriethoxysilane. These parameters influenced the enzymatic activity after immobilization. Gels prepared by addition of polyethylene oxide gave the best results and were used as monoliths in microreactors with two different geometries. The systems showed a high operational stability but a low effective enzyme activity. Enzyme leaching and a nonideal flow pattern may account for the low activity observed. This work is possibly the first one dealing with the immobilization of glucose 6‐phosphate dehydrogenase in silica‐based gels for its application in flow‐through microreactors.     

Glycerol‐3‐phosphate metabolism plays a role in stress response in the red alga Pyropia haitanensis

Glycerol‐3‐phosphate (G3P) has been suggested as a novel regulator of plant defense signaling, however, its role in algal resistance remains largely unknown. The glycerol kinase (also designated as NHO1) and NAD‐dependent G3P dehydrogenase (GPDH) are two key enzymes involved in the G3P biosynthesis. In our study, we cloned the full‐length cDNA of NHO1 (NHO1_Ph) and GPDH (GPDH_Ph) from the red alga Pyropia haitanensis (denoted as NHO1_Ph and GPDH_Ph) and examined their expression level under flagellin peptide 22 (flg22) stimulation or heat stress. We also measured the level of G3P and floridoside (a downstream product of G3P in P. haitanensis) under flg22 stimulation or heat stress. Both NHO1_Ph and GPDH_Ph shared high sequence identity and structural conservation with their orthologs from different species, especially from red algae. Phylogenetic analysis showed that NHO1s and GPDHs from red algae were closely related to those from animals. Under flg22 stimulation or heat stress, the expression levels of NHO1_Ph and GPDH_Ph were up‐regulated, G3P levels increased, and the contents of floridoside decreased. But the floridoside level increased in the recovery period after heat stress. Taken together, we found that G3P metabolism was associated with the flg22‐induced defense response and heat stress response in P. haitanensis, indicating the general conservation of defense response in angiosperms and algae. Furthermore, floridoside might also participate in the stress resistance of P. haitanensis.     

The role of sphingosine 1‐phosphate and its receptors in cardiovascular diseases

There are many different types of cardiovascular diseases, which impose a huge economic burden due to their extremely high mortality rates, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine 1‐phosphate (S1P) is a bioactive lipid mediator with paracrine and autocrine activities that acts through its cell surface S1P receptors (S1PRs) and intracellular signals. In the circulatory system, S1P is indispensable for both normal and disease conditions; however, there are very different views on its diverse roles, and its specific relevance to cardiovascular pathogenesis remains elusive. Here, we review the synthesis, release and functions of S1P, specifically detail the roles of S1P and S1PRs in some common cardiovascular diseases, and then address several controversial points, finally, we focus on the development of S1P‐based therapeutic approaches in cardiovascular diseases, such as the selective S1PR1 modulator amiselimod (MT‐1303) and the non‐selective S1PR1 and S1PR3 agonist fingolimod, which may provide valuable insights into potential therapeutic strategies for cardiovascular diseases.     

Glucose‐6‐phosphate dehydrogenase is posttranslationally regulated in the larvae of the freeze‐tolerant gall fly,Eurosta solidaginis,in response to freezing

The freeze‐tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) undergo substantial alterations to their molecular physiology during the winter including the production of elevated quantities of glycerol and sorbitol, which function as cryoprotectants to survive whole body freezing. Production of these cryoprotectants depends on cytosolic pools of nicotinamide adenine dinucleotide phosphate H (NADPH), a major source being the pentose phosphate pathway (PPP). Glucose‐6‐phosphate dehydrogenase (G6PDH) mediates the rate‐limiting and committed step of the PPP and therefore its molecular properties were explored in larvae sampled from control versus frozen states. G6PDH was purified from control (5°C) and frozen (?15°C) E. solidaginis larvae by a single‐step chromatography method utilizing 2′,5′‐ADP agarose and analyzed to determine its enzymatic parameters. Studies revealed a decrease in K_m for G6P in the frozen animals (to 50% of control values) suggesting an increased flux through the PPP. Immunoblotting of the purified enzyme showed differences in the relative extent of several posttranslational modifications, notably ubiquitination (95% decrease in frozen larvae), cysteine nitrosylation (61% decrease), threonine (4.1 fold increase), and serine phosphorylation (59% decrease). Together these data suggested that the increased flux through the PPP needed to generate NADPH for cryoprotectants synthesis is regulated, at least in part, through posttranslational alterations of G6PDH.     

High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA

The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum‐derived vesicles to Legionella‐containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4‐phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P‐binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.     

Sphingosine‐1‐phosphate (S1P) analog phytosphingosine‐1‐phosphate (P1P) improves the in vitro maturation efficiency of porcine oocytes via regulation of oxidative stress and apoptosis

Phytosphingosine‐1‐phosphate (P1P) is a signaling sphingolipid that regulates various physiological activities. However, little is known about the effect of P1P in the context of reproduction. Thus, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM). Here, we report the expression of S1PR1–3 among P1P receptors (S1PR1–4) in cumulus cells and oocytes. When P1P was administered at concentrations of 10, 50, 100, and 1,000 nM during IVM, the metaphase II rate was significantly increased in the 1,000 nM (1 μM) P1P treatment group. Maturation rate improvement by P1P supplementation was observed only in the presence of epidermal growth factor (EGF). Oocytes under the influence of P1P showed decreased intracellular reactive oxygen species levels but no significant differences in glutathione levels. In our molecular studies, P1P treatment upregulated gene expression involved in cumulus expansion (Has2 and EGF), antioxidant enzymes (SOD3 and Cat), and developmental competence (Oct4) while activating extracellular signal‐regulated kinase1/2 and Akt signaling. P1P treatment also influenced oocyte survival by shifting the ratio of Bcl‐2 to Bax while inactivating JNK signaling. We further demonstrated that oocytes matured with P1P displayed significantly higher developmental competence (cleavage and blastocyst [BL] formation rate) and greater BL quality (total cell number and the ratio of apoptotic cells) when activated via parthenogenetic activation (PA) and in vitro fertilization. Despite the low levels of endogenous P1P found in animals, exogenous P1P influenced animal reproduction, as shown by increased porcine oocyte maturation as well as preimplantation embryo development. This study and its findings are potentially relevant for both human and animal‐assisted reproduction.     

The effect of the sphingosine‐1‐phosphate analogue FTY720 on atrioventricular nodal tissue

The sphingosine‐1‐phosphate (S1P) receptor modulator, fingolimod (FTY720), has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular (AV) conduction block have been reported in some patients after the first dose. The underlying mechanism of this AV node conduction blockade is still not well‐understood. In this study, we hypothesize that expression of this particular arrhythmia might be related to a direct effect of FTY720 on AV node rather than a parasympathetic mimetic action. We, therefore, investigated the effect of FTY720 on AV nodal, using in vitro rat model preparation, under both basal as well as ischaemia/reperfusion conditions. We first look at the expression pattern of S1P receptors on the AV node using real‐time PCR. Although all three S1P receptor isoforms were expressed in AVN tissues, S1P1 receptor isoform expression level was higher than S1P2 and S1P3. The effect of 25 nM FTY720 on cycle length (CL) was subsequently studied via extracellular potentials recordings. FTY720 caused a mild to moderate prolongation in CL by an average 9% in AVN (n = 10, P < 0.05) preparations. We also show that FTY720 attenuated both ischaemia and reperfusion induced AVN rhythmic disturbance. To our knowledge, these remarkable findings have not been previously reported in the literature, and stress the importance for extensive monitoring period in certain cases, especially in patients taking concurrently AV node blocker agents.     

Structures of glyceraldehyde 3‐phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co‐infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng‐Ct co‐infections. Development of a safe, effective, and inexpensive dual therapy for Ng‐Ct co‐infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X‐ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high‐throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.