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Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water. In the gut flora, they activate azo pro-drugs, which are used for treatment of inflammatory bowel disease, releasing the active component 5-aminosalycilic acid. The bacterium P. aeruginosa has three azoreductase genes, paAzoR1, paAzoR2 and paAzoR3, which as recombinant enzymes have been shown to have different substrate specificities. The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form. We report here the characterization of the P. aeruginosa azoreductase enzymes, including determining their thermostability, cofactor preference and kinetic constants against a range of their favoured substrates. The expression levels of these enzymes during growth of P. aeruginosa are altered by the presence of azo substrates. It is shown that enzymes that were originally described as azoreductases, are likely to act as NADH quinone oxidoreductases. The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.

Site-2 proteases (S2Ps) constitute a large family of intramembrane cleaving proteases (I-CLiPs). S2Ps are metalloproteases and widely found in many species ranging from prokaryotes to higher eukaryotes. It is known that S2Ps participate in signal transduction related to stress response and lipid metabolism. In many systems, membrane-anchored precursor of transcription factor or suppressor protein of transcription factor are identified as the physiological substrates of S2Ps. The intramembrane proteolysis of the substrate results in release of the transcription factor from the membrane and expression of stress response genes. In Escherichia (E.) coli, an S2P homologue, RseP, is involved in the extracytoplasmic stress response. RseP cleaves the transmembrane sequence of the type II membrane protein RseA in cooperation with DegS protease and activates the transcription factor σ E . Similar to other site-2 proteolysis, the processing by RseP requires prior C-terminal truncation of RseA by DegS, but it remains unclear how RseP recognizes the truncated form of RseA. RseP possesses two PDZ modules in its periplasmic region and it is known that the PDZ modules of other proteins interact with the C-terminal tails of their ligands. In fact, it has recently been reported that the second PDZ module recognizes the C-terminal hydrophobic tail of RseA generated from the cleavage by DegS. However, it was also shown that point mutations to the first PDZ module have considerable effect on the substrate recognition mode of RseP. In this study, we produced and crystallized a soluble fragment of PDZ modules of RseP orthologue to analyze how the two PDZ modules are involved in the substrate recognition in the course of regulated intramembrane proteolysis. (propane-1,3-diamino)[N-(1-ferrocenyl methyl)]-4,4,6,6-tetrapyrrolidinocyclotriphosphaza triene (I) is a spirocyclic monoferrocenyl phosphazene derivative and it belongs to the space group P-1 with cell parameters a=13.475(2), b=15.041(3), c=19.666(9) Å and α=68.50(3)°, β=87.12(3)°, γ=66.32(2)°. The asymmetric unit of (I) contains two independent molecules. It has π-π contact between cyclopentadiene rings [centroid-centroid distance = 3.289(3) Å]. The C-H…N intermolecular hydrogen bonds[2] link the molecules, forming infinite one dimensional chains running approximately parallel to b axis. spiro (butane-1,4-diamino)[N,N'-bis(1-ferrocenyl-methyl)]-4,4,6,6-tetrachlorocyclotriphosphazatriene (II) is a spirocyclic bisferrocenyl phosphazene derivative including two ferrocenes and it belongs to the space group P-1 with cell parameters a=10.782(1), b=11.546(2), c=13.282(2) Å and α= 68.19(1)°, β=79.75(9)°, γ=88.62(1)°. It also has π-π contact between cyclopentadiene rings and the intramolecular C-H…N H bonds form a dimerization. Keywords: X-ray crystallography of proteins, molecular recognition, intramembrane proteolysis FA1-MS06-P09[1] Asmafiliz, N., Kilic, Z., Ozturk, A., Hokelek, T., Koc, L.Y., Acik, L., Kisa, O., Albay, A., Ustundag, Z., Solak, A.O., Inorg. Chem., 2009, 48 (21) Solu...

e23201 Background: Master transcriptional regulators (MTRs) are genes that are highly connected with many other genes and which can contribute to a specific cellular phenotype. The OncoMasTR panel, a set of 10 proliferation-linked MTRs and the key regulator of senescence CDKN2A, was identified in lymph node-negative breast cancer patients using publicly available data (Lanigan et al FEBS 2015 10.1111/febs.13354). Using two prognostic gene signatures and an experimentally defined ‘proliferative’ signature, highly concordant results for the top ranking sets of MTRs were found. One advantage of this process was the distillation of gene signatures into a discrete, mechanistically anchored biomarker set shown to be prognostically relevant, particularly in ER-positive patients, and with an ability to discriminate patients into low versus high risk of recurrence. Our aim here was to determine if the OncoMasTR panel was similarly useful in multiple other cancer types using publicly available transcriptomic data. Methods: The Gene Expression Omnibus was queried for datasets with relevant overall-, disease free- and/or recurrence free-survival information alongside microarray gene expression data. Survival analysis was conducted on 34 datasets using the R statistical environment package ‘survival’. Patient samples were scored based on median or tertile expression which were summed to give a final OncoMasTR RNA score, used to divide the patient samples into two groups (low and high risk) for analysis. Results: Using all 2,047 possible combinations of genes, a total of 23 datasets had significant prognostic associations. Following appropriate correction via multiple testing, this was reduced to 4 datasets representing bladder, lung and colorectal cancers, and multiple myeloma. Hazard ratios for the top 100 combinations in each data set were 1.8 – 2.1, 1.6 – 1.9, 1.1 – 1.3, 1.3 – 1.5 (standard error ±0.2, 0.1, 0.2, 0.1) respectively. All genes were found in the top ten combinations in all 4 datasets, and no specific combination was found to be prevalent across datasets, indicating that every MTR in the OncoMasTR panel contributes to its utility. Conclusions: The OncoMasTR panel is potentially prognostically useful in multiple cancer types.