No abstract

The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 Å), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYYbound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 Å apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.C-C coupling ͉ cyclopeptide

The extravasation of leukocytes from the peripheral blood stream to inflammatory sites is a key feature in the innate immune response to infection (1). Different chemoattractants (e.g. N-formylated peptides, C5a, interleukin-8, leukotriene B 4 , and platelet-activating factor) and chemokines induce leukocyte infiltration and activation through binding to G proteincoupled seven-transmembrane cell-surface receptors (2, 3). The chemoattractant-mediated dissociation of G␣ i2 from the G␤␥ subunit complex results in the activation of several downstream signaling effector enzymes that promote intracellular calcium mobilization, modifications in the metabolism of phosphoinositides, and activation of mitogen-activated protein kinases (4). The integration by the cell of the different chemoattractant-activated signaling pathways results in directed cell migration, recruitment of new receptors from the granules to the cell surface, release of proteolytic enzymes, production of large amounts of superoxide by the neutrophil NADPH oxidase, and increased gene transcription (5-8). The extent of the cellular response is dependent on the identity of the agonist and on the level of expression and desensitization of the receptors involved in the activation process (9).Two synthetic hexapeptides, Trp-Lys-Tyr-Met-Val-Met-NH 2 (WKYMVM)andTrp-Lys-Tyr-Met-Val-D-Met-NH 2 (WKYMVm), that stimulate phosphoinositide hydrolysis in myeloid cells were identified by screening a peptide library (10, 11). The D-methionine-containing hexapeptide (WKYMVm) was found to be a very potent activator of several leukocyte effector functions such as chemotaxis, mobilization of complement receptor-3, and activation of the NADPH oxidase (11). The peptide WKYMVm activates neutrophils through both the N-formyl peptide receptor (FPR) 1 and FPRL1 (N-formyl peptide receptor-like-1) (12, 13). The latter was originally cloned from human phagocytes by low-stringency hybridization of a cDNA library with the FPR cDNA sequence, and it was initially defined as an orphan receptor (14 -16). FPRL1 was later referred to as the LXA 4 receptor since it was shown to bind lipoxin A 4 with high affinity (17). In addition, several different peptides/proteins have been reported to stimulate this receptor. These include a leucine zipper-like domain of the HIV-1

SummaryUnder iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co-purification of FpvA with iron-free PaA and reported similar binding affinities of iron-free pyoverdin and ferric-pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron-free PaA and the FpvA receptor here reveals the existence of an FpvA±pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin-loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferricpyoverdin, it is shown that iron-free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric-pyoverdin displaces the bound iron-free pyoverdin on FpvA± PaA to form FpvA±PaA-Fe complexes. In vivo, the kinetics of formation of this FpvA±PaA-Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric-pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17-fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA-Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings.