Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate.     

Iridoid and caffeoyl phenylethanoid glycosides of the endangered carnivorous plant Pinguicula lusitanica L. (Lentibulariaceae)

This work reports for the first time the identification of the major compounds of Pinguicula lusitanica, an endangered carnivorous plant species, using minimal amounts of plant material. A methanol extract was prepared from in vitro cultured plantlets and analyzed by HPLC–SPE–NMR/HPLC–MS. Three iridoid and five caffeoyl phenylethanoid glycosides were identified. These groups of natural compounds were previously reported in the Lentibulariaceae family and have been used as chemotaxonomic markers in related families.     

Biological, thermal and photochemical transformation of 2-trifluoromethylphenol

This is the first report on the metabolism of trifluoromethyl aromatics in a thermophilic bacterium, Bacillus thermoleovorans A2. Enzymes of the phenol degradation pathway are induced when cultivating Bacillus thermoleovorans A2 on complex medium. Direct measurements of fluorinated xenobiotics in cell suspensions using 19F-NMR made it possible to follow quantitatively the biotransformation routes. During the biotransformation of 2-CF3-phenol by B. thermoleovorans A2, a fluorinated metabolite, 2-hydroxy-6-oxo-7,7,7,-trifluorohepta-2,4-dienoate (7-TFHOD), accumulated. This metabolite is transformed non-enzymatically when exposed to sunlight. The accumulation of 7-TFHOD as an intermediate in the 2-CF3-phenol pathway was rationalized by calculating molecular properties of a series of meta-cleavage products.     

β-Glucuronidase P408S,P415L mutations: evidence that both mutations combine to produce an MPS VII allele in certain Mexican patients

Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is an autosomal recessively inherited lysosomal storage disease caused by a deficiency in β-glucuronidase. We identified and studied a novel allele containing two C-to-T transitions resulting in P408S and P415L alterations, which is present in homozygous state in one Mexican patient and in heterozygous state in another. None of the previous reports describing mutations in the MPS VII gene include Mexican patients. Expression of either of the mutations individually showed only modest effects on the properties of the enzyme. However, expression of the doubly mutant allele resulted in markedly reduced activity and rapid degradation in an early biosynthetic compartment. Received: 13 December 1995 / Revised: 3 April 1996     

On Antarctoscyphus (Cnidaria, Hydrozoa), a new genus of antarctic hydroids and the description of two new species

A new genus of the hydroid family Sertulariidae is established. Several species formerly assigned to the genus Symplectoscyphus Marktanner-Turneretscher, 1890 are included. A review of those species is given, including their main features along with some remarks. Two species new to science are added; the material of those species was collected in the Scotia Sea by the Spanish Antarctic Expedition “Antártida 8611”. Present autecological data are discussed.     

Does a change in reactor loading rate affect activated sludge bioflocculation?

A collection of particles held together by different interparticle forces might eventually give rise to the formation of activated sludge flocs. This process is known as bioflocculation and is crucial for both conventional activated sludge systems and membrane bioreactors. Since industrial wastewater treatment plants generally face varying reactor loading rates due to varying production schemes in the facility, this paper investigates the impact of reactor loading rates on activated sludge bioflocculation. For this purpose, two reactors were initially operated at a nominal reactor loading rate (RLR) and afterwards changed to a high and low RLR. Based on the obtained results, it can be observed that sludge under low RLR conditions is prone to floc fragmentation due to an increase in water-soluble extracellular polymeric substances (EPS). The reactor under high RLR indicated increased floc erosion as a result of increased biomass concentration, which might imply more collisions between sludge flocs, releasing small sludge particles from the floc. In the high RLR reactor, no significant increase in EPS was observed. A distinction between the different (de)flocculation phenomena was made based on sludge volume index, effluent suspended solids and EPS data supplemented with microscopic image analysis.     

Deflavination and reconstitution of flavoproteins.

Flavoproteins are ubiquitous redox proteins that are involved in many biological processes. In the majority of flavoproteins, the flavin cofactor is tightly but noncovalently bound. Reversible dissociation of flavoproteins into apoprotein and flavin prosthetic group yields valuable insights in flavoprotein folding, function and mechanism. Replacement of the natural cofactor with artificial flavins has proved to be especially useful for the determination of the solvent accessibility, polarity, reaction stereochemistry and dynamic behaviour of flavoprotein active sites. In this review we summarize the advances made in the field of flavoprotein deflavination and reconstitution. Several sophisticated chromatographic procedures to either deflavinate or reconstitute the flavoprotein on a large scale are discussed. In a subset of flavoproteins, the flavin cofactor is covalently attached to the polypeptide chain. Studies from riboflavin-deficient expression systems and site-directed mutagenesis suggest that the flavinylation reaction is a post-translational, rather than a cotranslational, process. These genetic approaches have also provided insight into the mechanism of covalent flavinylation and the rationale for this atypical protein modification.     

Gene conversion as a mechanism for antigenic variation in trypanosomes

Expression of the gene coding for the trypanosome AnTat 1.1 surface antigen is linked to the duplicative transposition of a basic copy (BC) of this gene to an expression site. In two trypanosome clones successively derived from AnTat 1.1 (AnTat 1.10 and AnTat 1.1B) we found evidence that gene conversions are involved in the transformation of the AnTat 1.1 transposed element into the two new surface antigen coding sequences. Although the three resultant mRNAs--AnTat 1.1, 1.10, and 1.1B--are different, they still share large homologies. Two of them, AnTat 1.1 and 1.1B, code for surface coats that are indistinguishable by conventional serological techniques, whereas AnTat 1.10 has been found different by the same methods. The three genomic rearrangements involve two of the five members of the AnTat 1.1 gene family. These two members are both located in unstable telomeric regions similar to the expression site, each in a different orientation with respect to the DNA terminus. We have concluded that the duplicative transposition is achieved by a gene conversion that may affect variable lengths of the same silent genes, and that different members of the same surface antigen gene family can contribute to the diversification of the antigen repertoire.