scholarly journals The Bacterium Thermus thermophilus, Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

2003 ◽  
Vol 69 (6) ◽  
pp. 3272-3279 ◽  
Author(s):  
Nuno Empadinhas ◽  
Luciana Albuquerque ◽  
Anke Henne ◽  
Helena Santos ◽  
Milton S. da Costa
Keyword(s):  
Biosynthetic Pathway ◽  
Divalent Cations ◽  
Thermus Thermophilus ◽  
Maximal Activity ◽  
Thermophilic Bacterium ◽  
Hyperthermophilic Archaea ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Homologous Genes ◽  
Molecular Masses

ABSTRACT The biosynthetic pathway for the synthesis of the compatible solute α-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of α-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90�C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95�C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn2+ was most effective for MPGS, while Mn2+, Co2+, Mg2+, and to a lesser extent Ni2+ activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.

scholarly journals Characterization of the Biosynthetic Pathway of Glucosylglycerate in the Archaeon Methanococcoides burtonii

2006 ◽  
Vol 188 (3) ◽  
pp. 1022-1030 ◽  
Author(s):  
Joana Costa ◽  
Nuno Empadinhas ◽  
Luís Gonçalves ◽  
Pedro Lamosa ◽  
Helena Santos ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Biosynthetic Pathway ◽  
Thermus Thermophilus ◽  
Maximal Activity ◽  
Homologous Genes ◽  
Gene Coding ◽  
Dehalococcoides Ethenogenes ◽  
Recombinant Product ◽  
Organic Solute

ABSTRACT The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov ), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and “Dehalococcoides ethenogenes.” GpgS had maximal activity at 50°C. The maximal activity of GpgP was at 50°C with GPG as the substrate and at 60°C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.

scholarly journals The serum opacity reaction of Streptococcus pyogenes: general properties of the streptococcal factor and of the reaction in aged serum

1968 ◽  
Vol 66 (1) ◽  
pp. 37-47 ◽  
Author(s):  
M. J. Hill ◽  
Lewis W. Wannamaker
Keyword(s):  
Streptococcus Pyogenes ◽  
Divalent Cations ◽  
Horse Serum ◽  
Sodium Deoxycholate ◽  
Heat Stability ◽  
Maximal Activity ◽  
Free Enzyme ◽  
Ph Optimum ◽  
Serum Opacity Factor ◽  
High Concentrations

SUMMARYThe capacity of certain strains of Streptococcus pyogenes to produce opacity in aged horse serum has been studied. Cells from all stages of the growth cycle are able to produce opacity. Maximal activity is reached towards the end of the exponential phase of growth.Examination of cell fractions obtained by mechanical breakage and differential centrifugation suggested that the cell-bound activity is predominantly associated with the membrane fraction. Extraction with sodium deoxycholate yields a soluble fraction of high activity.There is considerable strain variation in heat stability of the serum opacity factor. Cell-bound activity is often quite resistant to heat, whereas extracted activity is less stable.Low concentrations of divalent cations have an activating effect, whereas high concentrations inhibit the serum opacity reaction. High concentrations of uni-valent cations are without effect on the cell-free enzyme but have an activating effect on the cell-bound enzyme.For both the cell-bound and the cell-free enzyme the pH optimum was 5·8.Although sensitive to trypsin and pepsin, the serum opacity factor appears to be resistant to streptococcal proteinase. Its activity is destroyed by formaldehyde and by periodate but is unaffected by a number of reducing agents.Pre-heating of the serum or the addition of iodoacetate did not affect the serum opacity reaction. The enhanced cholesterol esterification previously described with fresh serum appears to be a secondary reaction. Even when isolated by relatively gentle methods, α-lipoprotein serves as a substrate only in the presence of crystalline serum albumin.

scholarly journals Purification and kinetics of phenylpropanoid O-methyltransferase activities from Brassica oleracea

1989 ◽  
Vol 67 (11-12) ◽  
pp. 763-769 ◽  
Author(s):  
Emidio De Carolis ◽  
Ragai K. Ibrahim
Keyword(s):  
Competitive Inhibition ◽  
Divalent Cations ◽  
Product Inhibition ◽  
Affinity Column ◽  
Ph Optimum ◽  
Substrate Interaction ◽  
Interaction Kinetics ◽  
Molecular Masses ◽  
Inhibition Studies ◽  
Kinetics Of

Two phenylpropanoid O-methyltransferase isoforms were purified to homogeneity from young cabbage leaves. They catalyzed the meta-O-methylation of caffeic and 5-hydroxyferulic acids to ferulic and sinapic acids, respectively. Both isoforms I and II exhibited different elution patterns from a Mono Q column, distinct apparent pIs on chromatofocusing, different product ratios, and stability on adenosine–agarose affinity column. On the other hand, both isoforms had similar apparent molecular masses (42 kilodaltons) and a pH optimum of 7.6. They exhibited no requirement for divalent cations and were both irreversibly inhibited by iodoacetate. Substrate interaction kinetics of the more stable isoform I, using the 5-hydroxyferulic acid and S-adenosyl-L-methionine, gave converging lines. Product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and non-competitive inhibition between the phenylpropanoid substrate and its methylated product. The kinetic patterns are consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind and S-adenosyl-L-homocysteine is the last product released.Key words: phenylpropanoid O-methyltransferase, purification, isoforms, adenosine–agarose affinity chromatography, kinectic mechanism.

Metabolism of phosphoglycerides in E. coli IV. The positional specificity and properties of phospbolipase A

1969 ◽  
Vol 47 (12) ◽  
pp. 1125-1128 ◽  
Author(s):  
P. Proulx ◽  
C. K. Fung
Keyword(s):  
Sodium Lauryl Sulfate ◽  
Divalent Cations ◽  
Sodium Deoxycholate ◽  
Maximal Activity ◽  
Phospholipase A ◽  
Lauryl Sulfate ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
E Coli

The hydrolysis of labelled phosphatidylethanolamine by E. coli was studied in vitro. Phospholipase A, as detected by 32P-labelled lysophosphatidylethanolamine formation, had two pH optima, 5 and 8.4. On the other hand lysophosphofipase was active only in the alkaline range, had a pH optimum of 10, and was inhibited by high concentrations of either sodium deoxycholate or sodium lauryl sulfate. Phospholipase A required Ca2+ addition for maximal activity at both pH optima. Mg2+ also stimulated the activity but other divalent cations tested were slightly inhibitory or without effect. Sodium lauryl sulfate completely inhibited at pH 5. Experiments with singly and doubly labelled phosphatidylethanolamine indicated that phospholipase A1 activity was predominant at both acid and alkaline pH. Lower levels of phospholipase A2 were detectable only at alkaline pH.

scholarly journals Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27

FEBS Journal ◽  
2006 ◽  
Vol 273 (18) ◽  
pp. 4210-4218 ◽  
Author(s):  
Cornelia Schwarzenlander ◽  
Beate Averhoff
Keyword(s):  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Dna Transport

Biosynthesis of the Diguanosine Nucleotides. I. Purification and Properties of an Enzyme from Yolk Platelets of Brine Shrimp Embryos

1974 ◽  
Vol 52 (3) ◽  
pp. 231-240 ◽  
Author(s):  
A. H. Warner ◽  
P. C. Beers ◽  
F. L. Huang
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Artemia Salina ◽  
Temperature Optimum ◽  
Small Molecular Weight ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Purification And Properties

An enzyme that catalyzes the synthesis of P1P4-diguanosine 5′-tetraphosphate (Gp4G) has been isolated and purified from yolk platelets of encysted embryos of the brine shrimp, Artemia salina. The enzyme GTP:GTP guanylyltransferase (Gp4G synthetase) utilizes GTP as substrate, has a pH optimum of 5.9–6.0, a temperature optimum of 40–42 °C, and requires Mg2+ and dithiothreitol for optimal activity. The synthesis of Gp4G is inhibited markedly by pyrophosphate, whereas orthophosphate has no effect on the reaction. In the presence of GDP the enzyme also catalyzes the synthesis of P1,P3-diguanosine 5′-triphosphate (Gp3G), but the rate of synthesis is low compared with Gp4G synthesis and dependent upon other small molecular weight components of yolk platelets.

scholarly journals Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen MethanogenMethanobrevibacter milleraeSM9

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-6
Author(s):  
Yanli Zhang ◽  
Linley R. Schofield ◽  
Carrie Sang ◽  
Debjit Dey ◽  
Ron S. Ronimus
Keyword(s):  
Biosynthetic Pathway ◽  
Critical Role ◽  
Reduction Reaction ◽  
Coenzyme M ◽  
Purification And Characterization ◽  
The Third ◽  
Optimal Activity ◽  
Methyl Coenzyme M Reductase

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC fromMethanobrevibacter milleraeSM9 was cloned and expressed inEscherichia coliand biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen forα-ketoglutarate. Optimal activity was observed at pH 6.5. The apparentKMfor coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate theVmaxwas 93.9 μmol min−1 mg−1andkcatwas 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

scholarly journals Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Takeo Tomita ◽  
Hajime Matsushita ◽  
Ayako Yoshida ◽  
Saori Kosono ◽  
Minoru Yoshida ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Glutamate Dehydrogenase ◽  
Ternary Complex ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Regulatory Subunit ◽  
Binary Complex ◽  
Bacterial Cells ◽  
Adenine Phosphoribosyltransferase ◽  
Content Type

ABSTRACT Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells. IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

Extracellular alkaline phosphatase from marine bacteria: purification and properties of extracellular phosphatase from a marine Pseudomonas sp.

1980 ◽  
Vol 26 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Hiromi Kobori ◽  
Nobuo Taga
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Pseudomonas Sp ◽  
Purification And Properties ◽  
Extracellular Alkaline Phosphatase ◽  
Increased Pressure

Extracellular alkaline phosphatase produced by a marine Pseudomonas was purified to electrophoretic homogeneity. The molecular weight of the enzyme was estimated to be 100 000. The enzyme had maximal activity at pH 11.5. The enzyme was completely inhibited by 1 mM EDTA. However, divalent cations reversed the enzyme inhibition and their order of effectiveness on the reaction was Zn2+ > Ca2+ > Mn2+ > Mg2+ > Sr2+ > Co2+. The enzyme activity was affected by the species of anion whose order of effectiveness was demonstrated to follow the lyotrophic series, Cl− > Br− > NO3−> ClO4− > SCN−. The activity of phosphatase was accelerated linearly by increased pressure until up to 1000 atm (1 atm = 101.325 kPa), and the enzyme activity at 1000 atm was 3.2 times higher than that at 1 atm.

scholarly journals Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences

1998 ◽  
Vol 331 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Robert G. SPIRO ◽  
Vishnu D. BHOYROO
Keyword(s):  
Periodate Oxidation ◽  
Maximal Activity ◽  
Sialic Acid Residue ◽  
Lymphoid Tissues ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Selectin Ligands ◽  
Strict Requirement ◽  
Lectin Chromatography ◽  
Triton X

An enzyme which catalyses the transfer of sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcα2-3Galβ1-4GlcNAc (3´SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3´-sialyl-6´-sulpho-LacNAc [NeuAcα2-3Gal(6-SO4)β1-4GlcNAc] and 3´-sialyl-6´-sulpho-LewisX [NeuAcα2-3Gal(6-SO4)β1-4(Fucα1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an α2-3-linked sialic acid residue, although GlcNAc in 3´SLN can be substituted by Glc, and Galβ1-4GlcNAc can be replaced by Galβ1-3GlcNAc without loss of activity. The finding that 3´-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcα2-3Galβ1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3´SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra)bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3´SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12–25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 µM for PAPS, and 0.72 mM and 1.16 mM for 3´SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3´SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.

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