The Escherichia coli OxyR transcription factor senses H2O2 and is activated through the formation of an intramolecular disulfide bond. Here we present the crystal structures of the regulatory domain of OxyR in its reduced and oxidized forms, determined at 2.7 A and 2.3 A resolutions, respectively. In the reduced form, the two redox-active cysteines are separated by approximately 17 A. Disulfide bond formation in the oxidized form results in a significant structural change in the regulatory domain. The structural remodeling, which leads to different oligomeric associations, accounts for the redox-dependent switch in OxyR and provides a novel example of protein regulation by "fold editing" through a reversible disulfide bond formation within a folded domain.

The reactions of substituted phenolate anions with m-nitrophenyl, p-nitrophenyl, and 3,4-dinitrophenyl formates follow nonlinear Bransted-type correlations that might be taken as evidence for a change in the rate-limiting step of a reaction that proceeds through a tetrahedral addition intermediate. However, the correlation actually represents two different Bronsted lines that are defined by meta-and para-substituted phenolate anions and by meta-and para-substituted o-chlorophenolate anions. A concerted mechanism for both acetyl-and formyl-transfer reactions is supported by the absence of a detectable change in the Bronsted slope at ApK = 0 for the attacking and leaving phenolate anions within each class of Bronsted correlations. Regular increases in the dependence of log k on the pAa of the nucleophile with increasing pAa of the leaving group correspond to a positive interaction coefficient pxy = d/3lg/d(pA"uc) = d/3nuc/5(pAlg). The observation of two different Bronsted lines for the reactions of substituted phenolate anions with phenyl acetates is attributed to a steric effect that decreases the rate of reaction of substituted o-chlorophenolate anions by 25-50%. The reactions of metaand para-substituted phenolate and o-chlorophenolate anions with substituted phenyl acetate esters follow values of /3nlic = 0.53-0.66 and -j5lg = 0.50-0.63. The reactions of meta-and para-substituted phenolate anions with formate esters are ~103 times faster and follow smaller values of j6nuc = 0.43-0.64 and -/3|g = 0.31-0.48. However, the reactions of meta-and para-substituted o-chlorophenolate anions with the same formate esters follow larger values of /3nuc = 0.63-0.90 and -/3,g = 0.46-0.90. The large values of /3nuc and -/Slg for the reactions of substituted o-chlorophenolate anions with formate esters may arise from destabilization by the o-chloro group of a stacking interaction that is present in the transition state for reactions of formate esters, but not acetate esters.

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Purine catabolic pathway in Bacillus subtilis is consisted of more than 14 genes. Among these genes, pucL and pucM are required for uricase activity. While PucL is known to encode the uricase itself, the function of PucM is still unclear although this protein is also indispensable for uric acid decomposition. Here, we provide evidence that PucM, a transthyretin-related protein, functions to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Based on these results, we propose that transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as a hydroxyisourate hydrolase.

Previously, we identified annexin A4 (ANXA4) as a candidate substrate of caspase-3. Proteomic studies were performed to identify interacting proteins with a view to determining the roles of ANXA4. ANXA4 was found to interact with the p105. Subsequent studies revealed that ANXA4 interacts with NF-kappaB through the Rel homology domain of p50. Furthermore, the interaction is markedly increased by elevated Ca(2+) levels. NF-kappaB transcriptional activity assays demonstrated that ANXA4 suppresses NF-kappaB transcriptional activity in the resting state. Following treatment with TNF-alpha or PMA, ANXA4 also suppressed NF-kappaB transcriptional activity, which was upregulated significantly early after etoposide treatment. This difference may be due to the intracellular Ca(2+) level. Additionally, ANXA4 translocates to the nucleus together with p50, and imparts greater resistance to apoptotic stimulation by etoposide. Our results collectively indicate that ANXA4 differentially modulates the NF-kappaB signaling pathway, depending on its interactions with p50 and the intracellular Ca(2+) ion level.