Purification and characterization of FtsZ from the citrus canker pathogen Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xac) is the causative agent of citrus canker, a plant disease that significantly impacts citriculture. In earlier work, we showed that alkylated derivatives of gallic acid have antibacterial action against Xac and target both the cell division protein FtsZ and membrane integrity in Bacillus subtilis. Here, we have purified native XacFtsZ and characterized its GTP hydrolysis and polymerization properties. In a surprising manner, inhibition of XacFtsZ activity by alkyl gallates is not as strong as observed earlier with B. subtilis FtsZ. As the alkyl gallates efficiently permeabilize Xac membranes, we propose that this is the primary mode of antibacterial action of these compounds.

A simplified curcumin targets the membrane of Bacillus subtilis.

Curcumin is the main constituent of turmeric, a seasoning popularized around the world with Indian cuisine. Among the benefits attributed to curcumin are anti-inflammatory, antimicrobial, antitumoral, and chemopreventive effects. Besides, curcumin inhibits the growth of the gram-positive bacterium Bacillus subtilis. The anti-B. subtilis action happens by interference with the division protein FtsZ, an ancestral tubulin widespread in Bacteria. FtsZ forms protofilaments in a GTP-dependent manner, with the concomitant recruitment of essential factors to operate cell division. By stimulating the GTPase activity of FtsZ, curcumin destabilizes its function. Recently, curcumin was shown to promote membrane permeabilization in B. subtilis. Here, we used molecular simplification to dissect the functionalities of curcumin. A simplified form, in which a monocarbonyl group substituted the ß-diketone moiety, showed antibacterial action against gram-positive and gram-negative bacteria of clinical interest. The simplified curcumin also disrupted the divisional septum of B. subtilis; however, subsequent biochemical analysis did not support a direct action on FtsZ. Our results suggest that the simplified curcumin exerted its function mainly through membrane permeabilization, with disruption of the membrane potential necessary for FtsZ intra-cellular localization. Finally, we show here experimental evidence for the requirement of the ß-diketone group of curcumin for its interaction with FtsZ.

Antibacterial activity of monoacetylated alkyl gallates against Xanthomonas citri subsp. citri.

Asiatic citrus canker (ACC) is an incurable disease of citrus plants caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (X. citri). It affects all the commercially important citrus varieties in the major orange producing areas around the world. Control of the pathogen requires recurrent sprays of copper formulations that accumulate in soil and water reservoirs. Here, we describe the improvement of the alkyl gallates, which are potent anti-X. citri compounds, intended to be used as alternatives to copper in the control of ACC. Acetylation of alkyl gallates increased their lipophilicity, which resulted in potentiation of the antibacterial activity. X. citri exposed to the acetylated compounds exhibited increased cell length that is consistent with the disruption of the cell division apparatus. Finally, we show that inhibition of cell division is an indirect effect that seemed to be caused by membrane permeabilization, which is apparently the primary target of the acetylated alkyl gallates.

Metal-dependent SpoIIE oligomerization stabilizes FtsZ during asymmetric division in Bacillus subtilis.

SpoIIE is a bifunctional protein involved in asymmetric septum formation and in activation of the forespore compartment-specific transcription factor σF through dephosphorylation of SpoIIAA-P. The phosphatase activity of SpoIIE requires Mn2+ as a metal cofactor. Here, we show that the presence of a metal cofactor also influences SpoIIE oligomerization and asymmetric septum formation. Absence of Mn2+ from sporulation medium results in a delay of the formation of polar FtsZ-rings, similar to a spoIIE null mutant. We purified the entire cytoplasmic part of the SpoIIE protein, and show that the protein copurifies with bound metals. Metal binding both stimulates SpoIIE oligomerization, and results in the formation of larger oligomeric structures. The presence of SpoIIE oligomers reduces FtsZ GTP hydrolysis activity and stabilizes FtsZ polymers in a light scattering assay. Combined, these results indicate that metal binding is not just required for SpoIIE phosphatase activity but also is important for SpoIIE's role in asymmetric septum formation.

Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.

Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single- and double-mixing stopped-flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half-reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD. The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product-mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady-state rate equations derived for ping-pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme-product complex.

Turning a monocovalent flavoprotein into a bicovalent flavoprotein by structure-inspired mutagenesis.

A recently discovered class of bicovalent flavoproteins is an interesting group of enzymes because of their unusual cofactor binding mode, their open active sites and the bulky substrates they can accept. Through a sequence comparison study we have identified a conserved sequence region in bicovalent flavoproteins that is different from monocovalent flavoproteins. Based on this and the available structural information we have designed mutants of the prototype monocovalent flavoprotein, 6-hydroxy-d-nicotine oxidase (6HDNO), in order to introduce a second cofactor-protein linkage. Two amino acid replacements, namely histidine 130 to a cysteine and leucine 138 to a histidine, were sufficient to create a bicovalent 6HDNO. The introduced cysteine forms a covalent bond with FAD as found in natural bicovalent flavoproteins, while the second mutation was found to be essential to facilitate the formation of the cysteinyl linkage. This points to an important role of the introduced histidine in stabilizing a negative charge of the isoalloxazine ring during covalent flavinylation. The His130Cys/Leu138His 6HDNO is still active and shows a higher midpoint redox potential when compared to wild-type 6HDNO. This agrees well with the previous studies that have shown that bicovalent flavoenzymes have extremely high redox potentials.

Structure-based redesign of cofactor binding in putrescine oxidase.

Putrescine oxidase (PuO) from Rhodococcus erythropolis is a soluble homodimeric flavoprotein, which oxidizes small aliphatic diamines. In this study, we report the crystal structures and cofactor binding properties of wild-type and mutant enzymes. From a structural viewpoint, PuO closely resembles the sequence-related human monoamine oxidases A and B. This similarity is striking in the flavin-binding site even if PuO does not covalently bind the cofactor as do the monoamine oxidases. A remarkable conserved feature is the cis peptide conformation of the Tyr residue whose conformation is important for substrate recognition in the active site cavity. The structure of PuO in complex with the reaction product reveals that Glu324 is crucial in recognizing the terminal amino group of the diamine substrate and explains the narrow substrate specificity of the enzyme. The structural analysis also provides clues for identification of residues that are responsible for the competitive binding of ADP versus FAD (~50% of wild-type PuO monomers isolated are occupied by ADP instead of FAD). By replacing Pro15, which is part of the dinucleotide-binding domain, enzyme preparations were obtained that are almost 100% in the FAD-bound form. Furthermore, mutants have been designed and prepared that form a covalent 8α-S-cysteinyl-FAD linkage. These data provide new insights into the molecular basis for substrate recognition in amine oxidases and demonstrate that engineering of flavoenzymes to introduce covalent linkage with the cofactor is a possible route to develop more stable protein molecules, better suited for biocatalytic purposes.

Exploring the biocatalytic scope of a bacterial flavin-containing monooxygenase.

A bacterial flavin-containing monooxygenase (FMO), fused to phosphite dehydrogenase, has been used to explore its biocatalytic potential. The bifunctional biocatalyst could be expressed in high amounts in Escherichia coli and was able to oxidize indole and indole derivatives into a variety of indigo compounds. The monooxygenase also performs the sulfoxidation of a wide range of prochiral sulfides, showing moderate to good enantioselectivities in forming chiral sulfoxides.

Early vitrectomy and diagnostic testing in severe infectious posterior uveitis and endophthalmitis.

BACKGROUND: To determine the therapeutic and diagnostic value of vitreoretinal surgery in various types of severe viral retinitis and endogenous endophthalmitis. MATERIAL/METHODS: Pars plana vitrectomy with silicon-oil tamponade was performed on 12 eyes (12 patients) with severe viral retinitis and endogenous endophthalmitis. The mean age of the patients was 35.2 years (range, 18-56 years). Investigations consisted of chest radiography, RT 23, serology (for toxoplasmosis, cytomegalovirus, herpes simplex virus, and human immunodeficiency virus), and routine blood tests and urinalysis. Examination of vitreous samples consisted of polymerase chain reaction for cytomegalovirus, herpes simplex virus, and Mycobacterium tuberculosis, and cultures for fungi and bacteria. The results were analyzed 7 days and 6 months after surgery. RESULTS: Analysis of vitreous samples showed cytomegalovirus in 3 patients, herpes simplex virus in 2, tuberculosis in 2, and Candida albicans in 5. All patients had improvement in visual acuity and severity of clinical signs seven days after surgery and in 10 cases after long-term follow-up. Final visual acuity was compromised by chronic macular edema, postinflammatory macular scarring, optic atrophy, subcapsular cataract formation, and capsular opacification after cataract surgery. CONCLUSIONS: Modern laboratory tests are useful for quick and accurate diagnosis of atypical cases of severe infectious posterior uveitis. Early pars plana vitrectomy with silicon oil tamponade is valuable in diagnosing and treating severe infectious posterior uveitis and endophthalmitis.

Personality and music preferences: the influence of personality traits on preferences regarding musical elements.

The purpose of this scientific study was to determine how personality traits, as classified by Cattell, influence preferences regarding musical elements. The subject group consisted of 145 students, male and female, chosen at random from different Polish universities. For the purpose of determining their personality traits the participants completed the 16PF Questionnaire (Cattell, Saunders, & Stice, 1957; Russel & Karol, 1993), in its Polish adaptation by Choynowski (Nowakowska, 1970). The participants' musical preferences were determined by their completing a Questionnaire of Musical Preferences (specifically created for the purposes of this research), in which respondents indicated their favorite piece of music. Next, on the basis of the Questionnaire of Musical Preferences, a list of the works of music chosen by the participants was compiled. All pieces were collected on CDs and analyzed to separate out their basic musical elements. The statistical analysis shows that some personality traits: Liveliness (Factor F), Social Boldness (Factor H), Vigilance (Factor L), Openness to Change (Factor Q1), Extraversion (a general factor) have an influence on preferences regarding musical elements. Important in the subjects' musical preferences were found to be those musical elements having stimulative value and the ability to regulate the need for stimulation. These are: tempo, rhythm in relation to metrical basis, number of melodic themes, sound voluminosity, and meter.