Arabinose and protocatechuate catabolism genes are important for growth of Rhizobium leguminosarum biovar viciae in the pea rhizosphere

Background and aims: To form nitrogen-fixing nodules on pea roots, Rhizobium leguminosarum biovar viciae must be competitive in the rhizosphere. Our aim was to identify genes important for rhizosphere fitness. Methods: Signature-tagged mutants were screened using microarrays to identify mutants reduced for growth in pea rhizospheres. Candidate mutants were assessed relative to controls for growth in minimal medium, growth in pea rhizospheres and for infection of peas in mixed inoculants. Mutated genes were identified by DNA sequencing and confirmed by transduction. Results: Of 5508 signature-tagged mutants, microarrays implicated 50 as having decreased rhizosphere fitness. Growth tests identified six mutants with rhizosphere-specific phenotypes. The mutation in one of the genes (araE) was in an arabinose catabolism operon and blocked growth on arabinose. The mutation in another gene (pcaM), encoding a predicted solute binding protein for protocatechuate and hydroxybenzoate uptake, decreased growth on protocatechuate. Both mutants were decreased for nodule infection competitiveness with mixed inoculants, but nodulated peas normally when inoculated alone. Other mutants with similar phenotypes had mutations predicted to affect secondary metabolism. Conclusions: Catabolism of arabinose and protocatechuate in the pea rhizosphere is important for competitiveness of R.l. viciae. Other genes predicted to be involved in secondary metabolism are also important.

Síntese de derivados de ácidos fenólicos e estudo da sua actividade antioxidante

A peroxidação lipídica, além de causar sérios danos no corpo humano, é a principal causa da deterioração dos alimentos afectando a sua cor, aroma e valor nutricional, o que conduz a uma diminuição do seu ciclo de vida. O fenómeno de oxidação pode ser retardado com recurso a antioxidantes, de origem natural ou sintética, que inibam a formação de espécies reactivas, ou que reajam com estas, formando posteriormente radicais com menor grau de reactividade. Nesta dissertação procedeu-se à síntese e elucidação estrutural de novos antioxidantes, derivados dos ácidos 3,4-diidroxibenzóico (PCA) e 3,4-diidroxifenilacético (DOPAC), e à avaliação da sua actividade anti-radicalar e antioxidante. Os novos antioxidantes sintetizados foram caracterizados usando RMN de 1H e de 13C, FTIR e EM-IE. A avaliação da actividade antioxidante foi realizada com base no método do radical 2,2- difenil-1-picrilhidrazilo (DPPH·) e por técnicas electroquímicas (voltametria de impulso diferencial e voltametria cíclica). Os resultados obtidos permitiram concluir que a eficácia anti-radicalar (AE) é determinada por aspectos da estrutura molecular dos compostos, nomeadamente pela presença de grupos hidroxilo no anel aromático e também de grupos extensores relativamente à posição do grupo carboxílico. Os resultados permitiram verificar que o DOPAC apresenta a mais elevada eficiência anti-radicalar dos compostos em estudo, incluindo o trolox e o ácido gálhico (compostos de referência). Com base nos resultados obtidos concluiu-se que, um menor potencial de oxidação conduz a uma melhor actividade anti-radicalar dos compostos. De facto, verificou-se para o PCA e respectivos ésteres o maior potencial de oxidação e também a menor eficiência anti-radicalar. Em contrapartida, os antioxidantes com maior eficiência anti-radicalar, DOPAC, trolox e ácido gálhico, apresentaram menor potencial de oxidação.

Role of cis-cis muconic acid in the catalysis of Pseudomonas putida chlorocatechol 1,2-dioxygenase

Chlorocatechol 1,2-dioxygenase (1,2-CCD) is a non-heme iron protein involved in the intradiol cleavage of aromatic compounds that are recalcitrant to biodegradation. In particular, 1,2-CCD catalyzes the conversion of catechol and its halogenated derivatives to cis-cis muconic acid. In this study we describe a series of experiments concerning the interaction of chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp1,2-CCD) with cis-cis muconic acid. We used single-injection ITC to show that the reaction product inhibits enzyme kinetics. DSC and EPR measurements probed whether this was accomplished by a direct binding of the product to the enzyme active site. DSC shows that cis-cis muconic acid affects the thermal unfolding of the protein and allowed us to estimate a binding constant. Furthermore, EPR spectra of the Fe(III) center demonstrate that, upon product binding, a significant decrease in resonance intensity is observed, indicating that cis-cis muconic acid binds directly to the active site. Based on the increasing interest for understanding dioxygenases mechanism of action and, moreover, how to control such process, our data indicate that the product of the reaction does play a relevant role in the catalysis and should therefore be taken into account when one thinks about ways of regulating enzyme activity. (C) 2010 Elsevier B.V. All rights reserved.

Estudo da atividade antidermatofitica de Protocatecuatos contra T. rubrum e T. interdigitale

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Prospecção de substâncias anti histoplasma capsulatum nas formas planctônica e biofilme e análise proteômica

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Secondary metabolites from the wood bark of Durio zibethinus and Durio kutejensis

Phytochemical exploration of a wood bark extract from Durio zibethinus afforded two new triterpenoids, namely, methyl 27-O-trans-caffeoylcylicodiscate (1) and methyl 27-O-cis-caffeoylcylicodiscate (2), a new phenolic, 1,2-diarylpropane-3- ol (3), and seven known compounds, fraxidin, eucryphin, boehmenan, threo-carolignan E, (-)-(3R, 4S)-4-hydroxymellein, methyl protocatechuate, and (+)-(R)-de-O-methyllasiodiplodin (4). In addition, chemical analysis of a wood bark extract from Durio kutejensis yielded the new triterpenes 3 beta-O-trans-caffeoyl-2R-hydroxyolean-12-en-28-oic acid (5) and 3 beta-trans-caffeoyl-2R-hydroxytaraxest-12-en-28-oic acid (6) together with four known compounds, maslinic acid, arjunolic acid, 2,6-dimethoxy-p-benzoquinone, and fraxidin. The structures of all compounds were determined on the basis of spectroscopic data.