Previous studies have suggested that antibiotic vectorization by siderophores (iron chelators produced by bacteria) considerably increases the efficacy of such drugs. The siderophore serves as a vector: when the pathogen tries to take up iron via the siderophore, it also takes up the antibiotic. Catecholates are among the most common iron-chelating compounds used in synthetic siderophore-antibiotic conjugates. Using reverse transcription polymerase chain reaction and proteomic approaches, we showed that the presence of catecholate compounds in the medium of Pseudomonas aeruginosa led to strong activation of the transcription and expression of the outer membrane transporter PfeA, the ferri-enterobactin importer. Iron-55 uptake assays on bacteria with and without PfeA expression confirmed that catechol compounds imported iron into P. aeruginosa cells via PfeA. Uptake rates were between 0.3 x 10(3) and 2 x 10(3) Fe atoms/bacterium/min according to the used catechol siderophore in iron-restricted medium, and remained as high as 0.8 x 10(3) Fe atoms/bacterium/min for enterobactin, even in iron-rich medium. Reverse transcription polymerase chain reaction and proteomic approaches showed that in parallel to this switching on of PfeA expression, a repression of the expression of pyochelin (PCH) pathway genes (PCH being one of the two siderophores produced by P. aeruginosa for iron acquisition) was observed.
Title: Structure and action of a C-C bond cleaving alpha/beta-hydrolase involved in nicotine degradation Schleberger C, Sachelaru P, Brandsch R, Schulz GE Ref: Journal of Molecular Biology, 367:409, 2007 : PubMed
The enzyme 2,6-dihydroxy-pseudo-oxynicotine hydrolase from the nicotine-degradation pathway of Arthrobacter nicotinovorans was crystallized and the structure was determined by an X-ray diffraction analysis at 2.1 A resolution. The enzyme belongs to the alpha/beta-hydrolase family as derived from the chain-fold and from the presence of a catalytic triad with its oxyanion hole at the common position. This relationship assigns a pocket lined by the catalytic triad as the active center. The asymmetric unit contains two C(2)-symmetric dimer molecules, each adopting a specific conformation. One dimer forms a more spacious active center pocket and the other a smaller one, suggesting an induced-fit. All of the currently established C-C bond cleaving alpha/beta-hydrolases are from bacterial meta-cleavage pathways for the degradation of aromatic compounds and cover their active center with a 40 residue lid placed between two adjacent strands of the beta-sheet. In contrast, the reported enzyme shields its active center with a 110 residue N-terminal domain, which is absent in the meta-cleavage hydrolases. Since neither the substrate nor an analogue could be bound in the crystals, the substrate was modeled into the active center using the oxyanion hole as a geometric constraint. The model was supported by enzymatic activity data of 11 point mutants and by the two dimer conformations suggesting an induced-fit. Moreover, the model assigned a major role for the large N-terminal domain that is specific to the reported enzyme. The proposal is consistent with the known data for the meta-cleavage hydrolases although it differs in that the reaction does not release alkenes but a hetero-aromatic compound in a retro-Friedel-Crafts acylation. Because the hydrolytic water molecule can be assigned to a geometrically suitable site that can be occupied in the presence of the substrate, the catalytic triad may not form a covalent acyl-enzyme intermediate but merely support a direct hydrolysis.
