Activity of threonine deaminase and biosynthesis of avermectins in the culture of Streptomyces avermitilis

The influence of ammonium, threonine, isoleucine and valine on the activity of threonine deaminase and the biosynthesis of avermectins in the culture of two mutants of Streptomyces avermitilis, i.e. a sensitive one and a resistant one with respect to alpha-amino-beta-oxyvaleric acid, a threonine antimetabolite, was studied. It was shown that the synthesis of threonine deaminase was induced by threonine and valine in the mycelium of both the mutants. The level of threonine deaminase was higher in the mycelium of the antimetabolite resistant mutant. The antibiotic activity of the resistant mutant was lower while the relative content of the group B avermectins in the pool of the synthesized avermectins was higher than that in the culture of the sensitive mutant.     

Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli

Shifts in the sigmoidal kinetics of allosteric threonine deaminase promoted by isoleucine and valine binding control branched chain amino acid biosynthesis in Escherichia coli. A highly conserved alpha-helix in the C-terminal regulatory domain of the tetrameric enzyme was previously implicated in effector binding and feedback inhibition. Double (447, 451) and triple (447, 451, 454) alanine replacements for the conserved amino acids leucine 447, leucine 451, and leucine 454 in this region yield enzyme variants that show increased sigmoidality in steady-state kinetics, and which are less sensitive to the allosteric modifiers isoleucine and valine. Equilibrium binding studies using fluorescence, enzyme kinetic, and calorimetric approaches indicate that the enzyme variants possess reduced affinity for isoleucine and valine, and suggest that heterotropic ligands can bind to the same site to promote their different effects. The increase in sigmoidal kinetics for the mutants relative to wild-type threonine deaminase may be attributable to the elimination of L-threonine binding to the effector sites, which activates the wild-type enzyme. Enzyme kinetic data and isotherms for active site ligand binding to the mutants can be analyzed in terms of a simple two-state model to yield values for allosteric parameters that are consistent with previous estimates based on an expanded two-state model for homotropic cooperativity for threonine deaminase.     

Alpha-keto acid chain elongation reactions involved in the biosynthesis of coenzyme B (7-mercaptoheptanoyl threonine phosphate) in methanogenic Archaea

The biochemistry of the 13 steps involved in the conversion of alpha-ketoglutarate and acetylCoA to alpha-ketosuberate, a precursor to the coenzymes coenzyme B (7-mercapto heptanoylthreonine phosphate) and biotin, has been established in Methanosarcina thermophila. These series of reactions begin with the condensation of alpha-ketoglutarate and acetylCoA to form trans-homoaconitate. The trans-homoaconitate is then hydrated and dehydrated to cis-homoaconitate with (S)-homocitrate serving as an intermediate. Rehydration of the cis-homoaconitate produces (-)-threo-isohomocitrate [(2R,3S)-1-hydroxy-1,2, 4-butanetricarboxylic acid], which undergoes a NADP+-dependent oxidative decarboxylation to produce alpha-ketoadipate. The resulting alpha-ketoadipate then undergoes two consecutive sets of alpha-ketoacid chain elongation reactions to produce alpha-ketosuberate. In each of these sets of reactions, it has been shown that the homologues of cis-homoaconitate, homocitrate, and (-)-threo-isohomocitrate serve as intermediates. The protein product of the Methanococcus jannaschii MJ0503 gene aksA (AksA) was found to catalyze the condensation of alpha-ketoglutarate and acetylCoA to form trans-homoaconitate. This gene product also catalyzed the condensation of alpha-ketoadipate or alpha-ketopimelate with acetylCoA to form, respectively, the (R)-homocitrate homologues of (R)-2-hydroxy-1,2,5-pentanetricarboxylic acid and (R)-2-hydroxy-1,2, 6-hexanetricarboxylic acid. The alpha-ketosuberate resulting from this series of reactions then undergoes a nonoxidative decarboxylation to form 7-oxoheptanoic acid, a precursor to coenzyme B, and an oxidative decarboxylation to form pimelate, the precursor to biotin. Of the 13 intermediates in this pathway, eight have not previously been reported as occurring in biological systems.     

Cloning of nicotianamine synthase genes, novel genes involved in the biosynthesis of phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.     

Sulfonamide resistance in Streptococcus pyogenes is associated with differences in the amino acid sequence of its chromosomal dihydropteroate synthase

Sulfonamide resistance in recent isolates of Streptococcus pyogenes was found to be associated with alterations of the chromosomally encoded dihydropteroate synthase (DHPS). There were 111 different nucleotides (13.8%) in the genes found in susceptible and resistant isolates, respectively, resulting in 30 amino acid changes (11.3%). These substantial changes suggested the possibility of a foreign origin of the resistance gene, in parallel to what has already been found for sulfonamide resistance in Neisseria meningitidis. The gene encoding DHPS was linked to at least three other genes encoding enzymes of the folate pathway. These genes were in the order GTP cyclohydrolase, dihydropteroate synthase, dihydroneopterin aldolase, and hydroxymethyldihydropterin pyrophosphokinase. The nucleotide differences in genes from resistant and susceptible strains extended from the beginning of the GTP cyclohydrolase gene to the end of the gene encoding DHPS, an additional indication for gene transfer in the development of resistance. Kinetic measurements established different affinities for sulfathiazole for DHPS enzymes isolated from resistant and susceptible strains.     

Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene. The expression of an enzymatically active, wound-inducible tomato (Lycopersicon esculentum L. cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation. We constructed several deletion mutants of the gene, expressed these in E. coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC syntheses. Deletion of the COOH terminus through Arg429 results in complete inactivation of the enzyme. Deletion of 46-52 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme. The highly efficient, truncated ACC synthase was found to be a monomer of 52 +/- 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer. These results demonstrate that the non-conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.     

Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis

We previously reported the first cloning of a functional glycosaminoglycan synthase, the hyaluronan synthase (HAS) from Group A Streptococcus pyogenes (spHAS) (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184). Group A spHAS was unrelated to a putative Group C HA synthase reported by others (Lansing, M., Lellig, S., Mausolf, A., Martini, I. , Crescenzi, F., Oregon, M., and Prehm, P. (1993) Biochem. J. 289, 179-184). Here we report the isolation of a bona fide HA synthase gene from a highly encapsulated strain of Group C Streptococcus equisimilis. The encoded protein, designated seHAS, is 417 amino acids long (calculated molecular weight, 47,778; calculated pI, 9.1) and is the smallest member of the HAS family identified thus far. The enzyme migrates anomalously fast in SDS-polyacrylamide gel electrophoresis (approximately 42,000 Da). The seHAS protein shows no similarity (<2% identity) to the previously reported Group C gene, which is not an HA synthase. The seHAS and spHAS protein and coding sequences are 72 and 70% identical, respectively. seHAS is also similar to eukaryotic HAS1 (approximately 31% identical), HAS2 (approximately 28% identical), and HAS3 (28% identical). The deduced protein sequence of seHAS was confirmed by reactivity with a synthetic peptide antibody. Recombinant seHAS expressed in Escherichia coli was recovered in membranes as a major protein (approximately 10% of the total protein) and synthesized very large HA (Mr >7 x 10(6)) in the presence of UDP-GlcNAc and UDP-GlcA. The product contained equimolar amounts of both sugars and was degraded by the specific Streptomyces hyaluronidase. Comparison of the two recombinant streptococcal enzymes in isolated membranes showed that seHAS and spHAS are essentially identical in the steady-state size distribution of HA chains they synthesize, but seHAS has an intrinsic 2-fold faster rate of chain elongation (Vmax) than spHAS. seHAS is the most active HA synthase identified thus far; it polymerizes HA at an average rate of 160 monosaccharides/s. The two bacterial HA synthase genes may have arisen from a common ancient gene shared with the early evolving vertebrates.     

Leukotriene A4 hydrolase and leukotriene C4 synthase activities in human chondrocytes: transcellular biosynthesis of Leukotrienes during granulocyte-chondrocyte interaction

OBJECTIVE: To investigate the cooperation of chondrocytes and polymorphonuclear cells (PMN) in the biosynthesis of leukotrienes (LT). METHODS: PMN, resting and interleukin-1beta-stimulated cultured human chondrocytes, and mixtures of both cell types were incubated with A23187 and/or 14C-arachidonic acid (14C-AA). To explore the presence of LTC4 synthase and LTA4 hydrolase, the chondrocytes were incubated with authentic LTA4. Eicosanoids were analyzed using high performance liquid chromatography techniques. RESULTS: Chondrocytes formed only prostaglandin E2 and minor amounts of 15-HETE and 11-HETE, the production of all of which was inhibited by 1 microM indomethacin. Incubation of PMN and chondrocytes produced more LTC4 from endogenous and exogenous AA, and more LTB4 from endogenous AA, than incubation of PMN alone, which was consistent with the presence of LTC4 synthase and LTA4 hydrolase activities in chondrocytes. Chondrocytes also slightly increased the level of PMN production of all 5-lipoxygenase (5-LO)-derived products from endogenous AA. CONCLUSION: Human chondrocytes form eicosanoids from AA only by the cyclooxygenase pathway. Chondrocytes cooperate in the transcellular biosynthesis of LT since they possess LTA4 hydrolase and LTC4 synthase activities and increase metabolism by the 5-LO pathway in PMN.     

Biological properties of Streptococcus bovis bacteriophages isolated from lysogenic cultures and sheep rumen

Biological characteristics of eleven phages for Streptococcus bovis were investigated; seven phage were isolated from ovine rumen and four were virulent mutants of temperate phages of lysogenic cultures. The phages had many properties in common: similar morphology of negative colonies, the identical spectrum of lytic action, related antigens, absolute or high requirement of calcium ions, thermolability, and inactivation by the content of the rumen. Their susceptibility to the inactivating action of acetic acid, urea and temperature was however different. Chloroform and phenol may be used during purification and conservation of the phages.     

Characterization of the L-malate permease gene (maeP) of Streptococcus bovis ATCC 15352

A gene which was shown to be cotranscribed with the NAD+-dependent malic enzyme gene (maeE) of Streptococcus bovis ATCC 15352 was revealed to encode L-malate-specific permease (MaeP), which showed high activity at low pHs (pH 5.1 to 5.9). MaeP was strongly inhibited by the ionophores nigericin and valinomycin.     

Riboflavin biosynthesis in Saccharomyces cerevisiae. Cloning, characterization, and expression of the RIB5 gene encoding riboflavin synthase

Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity. Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity. Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus. Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity by a simple procedure. The specific activity of the enzyme was enriched 8500-fold. The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein. Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.     

Chronic morphine increases biosynthesis of nitric oxide synthase in the rat spinal cord

The present study was undertaken to determine the influence of chronic morphine treatment on the biosynthesis of nitric oxide synthase (NOS) in the rat spinal cord using in situ hybridization and immunohistochemical methods. Repeated administration of morphine (20-100 mg/kg/day; 10 days) increased the NOS mRNA level in laminae I-IV and X 3 h after the last injection. That effect was accompanied by an increase in both the number of NOS-positive cells (24 h) and the optical density of NOS-immunoreactivity (3 and 24 h). The results indicate that repeated morphine administration increases NOS biosynthesis in the rat spinal cord, which may reflect adaptive changes accounting for development of opiate tolerance and dependence.     

Serum erythrocyte and leukocyte adenosine deaminase activities in patients with vivax malaria in Turkey

Adenosine deaminase (ADA) activities in serum samples, erythrocytes, leukocytes and plasma hemoglobin concentrations were investigated in 50 patients with vivax malaria and compared with control group. ADA activity was determined by Bertholet reaction. Student's t-test and correlation analyses methods were used for statistical analyses. Serum ADA activity in patients with vivax malaria 49.20 +/- 29.02 IU/I, in control 21.15 +/- 8.04 IU/I (p = 0.005), erythrocyte ADA activity in patients 2.91 +/- 1.23 U/gr Hb, in control 1.65 +/- 0.59 U/gr (p = 0.001), leukocyte specific ADA activity in patients 26.23 +/- 20.21 U/mg protein, in control 25.84 +/- 9.19 U/gr Hb were determined (P > 0.05). Plasma hemoglobin concentration in patients 29.25 +/- 28.10 ml/dl, in control 9.80 +/- 13.14 mg/dl were also determined. There is no significant correlation among mentioned parameters. Erythrocyte purine salvage pathway is accelerated by Plasmodium to provide preformed purine source which can not be synthesized by Plasmodium to provide preformed purine source which can correlation between plasma hemoglobin concentration and serum ADA activity suggests that increased serum ADA activity may develop secondarily to the disease independently from the hemolyses. No higher ADA activity level than expected value of leukocytes may reflect immunosuppression of leukocytes.     

Amelioration of virulent Babesia bovis infection in calves by administration of the nitric oxide synthase inhibitor aminoguanidine

The expression of synaptosomal-associated protein (SNAP-25), neural growth-associated protein (GAP-43) and neural cell adhesion molecule (NCAM) were studied in mouse olfactory cells and axons for 2 weeks following unilateral bulbectomy. The olfactory cells and axons in the control olfactory epithelium were positive for SNAP-25 but levels decreased in the atrophic olfactory epithelium 3 days after bulbectomy. There was no expression of SNAP-25 in the olfactory epithelium on the bulbectomy side 7 days after bulbectomy, indicating that this protein may be a good marker for the degeneration of olfactory cells. The expression of NCAM was still found in the atrophic olfactory epithelium at 7 days after bulbectomy, while the expression of NCAM in the olfactory epithelium of the bulbectomy side was stronger than that on the control side at 14 days after bulbectomy. The expression of GAP-43 in the olfactory axonal bundles of the bulbectomy side at 3 and 4 days after bulbectomy was stronger than that on the control side. These results suggest that upregulation of NCAM may be related to the regeneration of the olfactory cells, with upregulation of GAP-43 probably playing a role in axonal regeneration after bulbectomy.     

Mechanistic studies of the biosynthesis of paratose: purification and characterization of CDP-paratose synthase

The 3,6-dideoxyhexoses can be found in the cell wall lipopolysaccharide of Gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. All naturally occurring 3,6-dideoxyhexoses, with colitose as the only exception, are biosynthesized via a complex pathway that begins with CDP-d-glucose. Included in this pathway is CDP-paratose synthase, an essential enzyme in the formation of the 3,6-dideoxy sugars, CDP-paratose and CDP-tyvelose. Recently, the gene encoding CDP-paratose synthase in Salmonella typhi, rfbS, has been identified and sequenced [Verma, N., and Reeves, P. (1989) J. Bacteriol. 171, 5694-5701]. On the basis of this information, we have amplified the rfbS gene by polymerase chain reaction (PCR) from S. typhi and cloned this gene into a pET-24(+) vector. Expression and purification of CDP-paratose synthase have allowed us to fully characterize the catalytic properties of this enzyme, which is a homodimeric protein with a preference for NADPH over NADH. It catalyzes the stereospecific hydride transfer of the pro-S hydrogen from the C-4' position of the reduced coenzyme to C-4 of the substrate, CDP-3,6-dideoxy-D-glycero-D-glycero-4-hexulose. The overall equilibrium of this catalysis greatly favors the formation of the reduced sugar product and the oxidized coenzyme. Interestingly, this enzyme also exhibits a high affinity for NADPH with a much smaller dissociation constant (Kia) of 0.005 +/- 0.002 microM compared to the Km of 26 +/- 8 microM for NADPH. While this unusual property complicated the interpretation of the kinetic data, the kinetic mechanism of CDP-paratose synthase as explored by the combination of bisubstrate kinetic analysis, product inhibition studies, and dead-end competitive inhibition studies is most consistent with a Theorell-Chance mechanism. The present study on CDP-paratose synthase, a likely new member of the short-chain dehydrogenase family, represents the first detailed characterization of this type of ketohexose reductase, many of which may share similar properties with CDP-paratose synthase.     

Regulation of early cholesterol biosynthesis in rat liver: effects of sterols, bile acids, lovastatin, and BM 15.766 on 3-hydroxy-3-methylglutaryl coenzyme A synthase and acetoacetyl coenzyme A thiolase activities

BACKGROUND: Sclerosing peritonitis (SP) is a rare but serious complication of peritoneal dialysis (PD). Small-bowel obstruction (SBO) due to encapsulation, dense adhesions, or mural fibrous is characteristic, often associated with peritonitis. The aim of the study was to determine the incidence, clinical features, effect of duration of dialysis, and other possible aetiological factors in severe SP. METHODS: All dialysis units in Australia were surveyed for possible cases up to 1994. Patients were included if there was either surgical or radiological evidence of sclerosing encapsulating peritonitis or SBO with tanned or thickened peritoneum in the absence of other causes of SBO. RESULTS: Fifty-four patients were analysed. The duration of continuous PD was mean 52 +/- 30 months, median 48 months and range 8-127 months. Nineteen cases were diagnosed between 1980 and 1989 and 35 between 1990 and 1994, giving mean annual incidences 1.9 and 4.2 per 1000 PD periods respectively. The overall prevalence was 0.7%, which increased progressively with the duration of PD being 1.9, 6.4, 10.8, and 19.4% for patients on dialysis for > 2, 5, 6 and 8 years respectively. Sclerosing encapsulating peritonitis was diagnosed in 87% of cases, SBO in 92%, and haemoperitoneum in 8%. Peritoneal calcification was present in seven cases, all of which had been on PD > 7 years. Peritonitis was associated with 38% of cases with fungal infection in 7%. Treatment with immunosuppression in five patients appeared to result in a favourable outcome in three. The mortality rate was 56%. CONCLUSION: Severe sclerosing peritonitis is a serious complication of peritoneal dialysis and there is a time dependent increase on CAPD.     

Relationship between intracellular phosphate, proton motive force, and rate of nongrowth energy dissipation (energy spilling) in Streptococcus bovis JB1

When the rate of glucose addition to nongrowing Streptococcus bovis cell suspensions was increased, the fermentation was homolactic, fructose-1,6-diphosphate (FDP) increased, intracellular inorganic phosphate (P(i)) declined, and the energy-spilling rate increased. ATP and ADP were not significantly affected by glucose consumption rate, but the decrease in P(i) was sufficient to cause an increase in the free energy of ATP hydrolysis (delta G'p). The increase in delta G'p was correlated with an increase in proton motive force (delta p). S. bovis continuous cultures (dilution rate of 0.65 h-1) that were provided with ammonia as the sole nitrogen source also had high rates of lactate production and energy spilling. When Trypticase was added as a source of amino acids, lactate production decreased; a greater fraction of the glucose was converted to acetate, formate, and ethanol; and the energy-spilling rate decreased. Trypticase also caused a decrease in FDP, an increase in P(i), and a decrease in delta p. The change in delta p could be explained by P(i)-dependent changes in the delta G'p. When P(i) declined, delta G'p and delta p increased. The ratio of delta G'p to delta p (millivolt per millivolt) was always high (> 4) at low rates of energy spilling but declined when the energy-spilling rate increased. Based on these results, it appears that delta p and the energy-spilling rate are responsive to fluctuations in the intracellular P(i) concentration.