Caracterización de Geranil-CoA y 3-Metilcrotonil-CoA Carboxilasas de Pseudomonas aeruginosa involucradas en la degradación de leucina e isoprenoides acíclicos

Abstract

Hydrocarbon (n-alkanes) degradation by microorganisms occurs via the fatty acids oxidation enzymes; however, if the hydrocarbon has a branched-chain structure different catabolic pathways are necessary. The isoprenoid citronellol and the amino acid leucine are branched-chain compounds. Leucine and isovalerate catabolic pathway used by Pseudomonas aeruginosa to produce acetoacetate and acetyl-CoA as final products is similar to the citronellol catabolic pathway in the same bacteria, and share analogous enzymes in both pathways. Moreover, 3-methylcrotonil-CoA (MC-CoA), the end product of the citronellol degradation, is taken by the leucine catabolic enzymes to produce acetoacetate and acetyl- CoA. The leucine degradation pathway complements the acyclic isoprenoids catabolism and drives both of them to acetoacetate and acetyl-CoA. The geranyl-CoA carboxylase (GCCase) enzyme from P. citronellolis, in the acyclic isoprenoid catabolism, and the 3-methylcrotonil- CoA carboxylase (MCCase) in the leucine and isovalerate catabolism, both have been considered as key enzymes in their respective pathways. These enzymes are encoded by the atuCF and liuDB genes, respectively, in the P. aeruginosa PAO1 chromosome. Furthermore, there is evidence indicating that both enzymes probably have activity in the two pathways. The proteins were expressed in Escherichia coli as recombinant proteins, before affinity- chromatography purification, a denaturalization and renaturalization procedures were undertaken to obtain functional enzymes. The two carboxylases showed pH and temperature optima of 8.5 and 37oC, respectively.

In the present work we investigated the down and up-regulation of the function of the gene coding for the subunit f of the eukaryotic initiation factor 3 (eIF3f) and evaluated its cellular effect. The eIF3f gene plays an important role in the initiation of translation (Benne y Hershey 1978). We originally found that the transcriptional expression of the common bean (Phaseolus vulgaris) eIF3f gene was related to cell division. Laboratory results showed that expression of the eIF3f bean gene in meristematic tobacco cells TBY-2 is circumscribed to the G2/M transition of the cell cycle, and that it is essential for the progression of the cell cycle. Recent results suggest that eIF3f is a negative regulator of translation and has a role in apoptotic signaling (Shi et al. 2006). Therefore, it was interesting to investigate if deregulation of eIF3f expression had an effect on human cell proliferation. We investigated the effect of ectopic expression and silencing of eIF3f gene on cellular growth of a human lung adenocarcinome (cell line A549) with the objective of gathering evidence that indicated its importance in cell division and its possible use in gene therapy against cancer. We found that in quiescent cultures, arrested in G0 by serum limitation, the levels of expression of eIF3f are lowered, compared to proliferating cultures. To determine the expression profile of human eIF3f gene during the cell cycle, A549 cells were synchronized and samples were taken after arrest release (mitotic index, RNA and protein). Expression profiles of both the mRNA and the protein of human eIF3f showed the same cyclic outline. La degradación de hidrocarburos (n-alcanos) mediada por microorganismos se efectúa mediante las enzimas de la oxidación de ácidos grasos, sin embargo, se requiere de rutas catabólicas alternas cuando estos hidrocarburos presentan ramificaciones en su estructura.