Salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans: crystal structure of a peculiar ring-cleaving dioxygenase

The crystallographic structure of salicylate 1,2-dioxygenase (SDO), a new ring fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans BN12, which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring fission mechanism, has been solved by molecular replacement techniques and refined at 2.9 Å resolution (R_free 26.1%; R-factor 19.3%). SDO is a homo-tetramer member of type III extradiol-type dioxygenases with a subunit topology characteristic of the bicupin β-barrel folds. The catalytic center contains a mononuclear iron(II) ion coordinated to three histidine residues (His119, His121, and His160), located within the N-terminal domain in a solvent-accessible pocket. SDO is markedly different from the known gentisate 1,2-dioxygenases (GDO) or 1-hydroxy-2-naphthoate dioxygenase because of its unique ability to oxidatively cleave numerous salicylates, gentisates and 1-hydroxy-2-naphthoate with high catalytic efficiency. The comparison of the structure and substrate specificity for a series of different substrates with the corresponding data for several GDOs and the docking of salicylates/gentisates in the active site of SDO, allowed the identification of several active site residues responsible for differences of substrate specificity. In particular, a more defined electron density of the N-terminal region allowed the discovery of a novel structure fragment in SDO previously unobserved in GDO. This region contributes several residues to the active site that influence substrate specificity for both of these enzymes. Implications on the catalytic mechanism are discussed.     

Characterization of adjacent E-box and nuclear factor 1-like DNA binding sequence in the human CYP1A2 promoter

To better understand the molecular mechanisms of cytochrome P450 1A2 (CYP1A2) regulation, we have characterized a region of the promoter (+3 to -176) that contains a single E-box and an adjacent nuclear factor 1 (NF1)-like DNA binding site. The E-box was shown to specifically bind nuclear proteins that were recognized by antibodies against upstream stimulatory factor (USF) 1 and 2. Comparison of NF1 binding proteins in HepG2 cells and primary cultures of rat hepatocytes revealed different patterns of DNA-protein complexes, all of which were recognized by a general NF1 antibody. Mutations of the E-box resulted in substantial reduction of promoter activity in either primary hepatocytes or HepG2 cells regardless of the presence in the reporter constructs of other CYP1A2 regulatory elements, such as the hepatic nuclear factor 1 (HNF-1) binding site. In contrast, reporter gene activity of the promoter construct harboring the mutated NF1-like binding site was affected by upstream sequences when transfected into HepG2 cells, but not in primary hepatocytes. We conclude that both USF proteins and different isoforms of NF1 contribute to the constitutive expression of CYP1A2.     

Sex, secondary compounds and asymmetry. Effects on plant–herbivore interaction in a dioecious shrub

We analysed the links between herbivory, anthraquinone content and developmental instability of leaves in Rhamnus alpinus, taking into account possible effects of sexual dimorphism. The amount of leaf loss caused by herbivores averaged 3%, rarely exceeding 25%. Leaf losses were evenly distributed in the shrubs, with highest variability among leaves of the same shoot, thus hiding possible shrub, sex or population effects. This pattern of herbivory implies a shifting of caterpillars from one leaf to another before consuming all readily available material. We suggest that this behaviour might be triggered by a short-term change in leaf palatability by means of an increase in the production of secondary compounds. Supporting this hypothesis, we have found a higher anthraquinone content in damaged leaves compared with undamaged ones. The leaves of male plants exhibited a higher concentration of anthraquinones than those of females, which contrasts with classic hypotheses. We relate this to the lower rate of biomass increase in males, which should allow them to allocate more resources to defence. Leaves showed fluctuating asymmetry (FA), but we did not find any relationship between the degree of asymmetry and sex, herbivory or anthraquinone content at any level considered. Therefore, FA cannot be considered as an indicator of susceptibility to damage by herbivores or of the ability to induce the production of defensive compounds in R. alpinus.     

Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues

Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.     

Aflatoxin M1 effects on Xenopus laevis development

BACKGROUND: The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. METHODS: Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. RESULTS: A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. CONCLUSIONS: Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.     

Modification by glucose of the flocculent phenotype of a <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> wine strain

We have evaluated the induction of the flocculent phenotype of Kloeckera apiculata by glucose mc1 and propose a pathway involved in carbohydrate flocculation induction. Pulses of glucose were given to cells growing in glucose-poor medium (2 g l(-1)) and the flocculation percentage was measured. To elucidate the mechanism involved in flocculation induction, cycloheximide was injected into the cultures 120 min before the glucose pulse. 2,4-Dinitrophenol or cAMP was added to the media instead, or simultaneously with glucose, while a protein kinase A (PKA) inhibitor was added 30 min before the glucose pulse. With 20 and 50 g l(-1) glucose pulse, the yeast flocculation percentage arises to 55 and 65%, respectively. The quantity of proteins and the reflocculating capacity of a lectinic protein extract from the yeast cell wall increase as the concentration of glucose pulse was higher. Cycloheximide prevented the glucose-induced flocculation, while cAMP or 2,4-dinitrophenol increased it 4- and 5-fold, respectively. PKA inhibitor completely prevented the glucose induction flocculation. The flocculent phenotype of K. apiculata mc1 was induced by glucose and the mechanism seems to imply de novo protein (lectin) synthesis via the PKA transduction pathway. This work contributes to the elucidation of the mechanism involved in flocculation induction by glucose of a non-Saccharomyces wine yeast, K. apiculata, which has not been reported. The induction of flocculation by glucose could be a biotechnological tool for the early removal of the indigenous microorganisms from the grape must before the inoculation of a selected starter strain to conduct the alcohol fermentation.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

Resistance to glyphosate in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> as result of pre-selective mutations

Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive (G^s) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally the growth of rare cells resistant (G^r) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10⁻⁷ to 3.6 × 10⁻⁷ mutants per cell per generation in two strains of M. aeruginosa; the frequency of the G^r allele ranged from 6.14 × 10⁻⁴ to 6.54 × 10⁻⁴. The G^r mutants are slightly elliptical in outline, whereas the G^s cells are spherical. Since G^r mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via G^r clone selection.