Context-dependent remodeling of Rad51-DNA complexes by Srs2 is mediated by a specific protein-protein interaction.

The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3' ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s(-1), 25 °C), (ii) ATP hydrolysis (kATP = 70 s(-1), 25 °C), (iii) Phosphate release (kPi = 16 s(-1), 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s(-1), 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe(ox)(ADP)2 protein and the reduced MoFe protein.

Specific chlorination of isoquinolines by a fungal flavin-dependent halogenase.

Rdc2 is the first flavin-dependent halogenase identified from fungi. Based on the reported structure of the bacterial halogenase CmlS, we have built a homology model for Rdc2. The model suggests an open substrate binding site that is capable of binding the natural substrate, monocillin II, and possibly other molecules such as 4-hydroxyisoquinoline (1) and 6-hydroxyisoquinoline (2). In vitro and in vivo halogenation experiments confirmed that 1 and 2 can be halogenated at the position ortho to the hydroxyl group, leading to the synthesis of the chlorinated isoquinolines 1a and 2a, respectively, which further expands the spectrum of identified substrates of Rdc2. This work revealed that Rdc2 is a useful biocatalyst for the synthesis of various halogenated compounds.

Cyclooxygenase 2 pathway and its therapeutic inhibition in superantigen-induced toxic shock.

Bacterial superantigens are a family of exotoxins that are the most potent T-cell activators known. Because of their ability to induce strong immune activation, superantigens have been implicated in a variety of diseases ranging from self-limiting food poisoning to more severe toxic shock syndrome (TSS) and have the potential to be used as agents of bioterrorism. Nonetheless, the precise molecular mechanisms by which T-cell activation by superantigens lead to acute systemic inflammatory response, multiple organ dysfunction, and ultimately death are unclear. Inadequate understanding of the pathogenesis has resulted in lack of development of effective therapy for superantigen-induced TSS. To fill these deficiencies, we systematically dissected the molecular pathogenesis of superantigen-induced TSS using the humanized human leukocyte antigen-DR3 transgenic mouse model by microarray-based gene expression profiling. Splenic expression of prostaglandin-endoperoxide synthase 2 (PTGS-2; also called cyclooxygenase 2 or COX-2) gene was increased by several hundred folds shortly after systemic superantigen (staphylococcal enterotoxin B [SEB]) exposure. In addition, expressions of several genes associated with eicosanoid pathway were significantly modulated by SEB, as analyzed by dedicated software. Given the importance of the COX-2 pathway in inflammation, we examined whether therapeutic inhibition of COX-2 by a highly selective inhibitor, CAY10404, could be beneficial. Our studies showed that i.p. administration of CAY10404 (50 mg/kg) immediately after challenge with 10 microg of SEB was unable to inhibit SEB-induced in vivo cytokine/chemokine production or T-cell activation/proliferation and did not prevent superantigen-associated thymocyte apoptosis.