Crystal structure of the monocupin ring-cleaving dioxygenase 5-nitrosalicylate 1,2-dioxygenase from Bradyrhizobium sp.

5-Nitrosalicylate 1,2-dioxygenase (5NSDO) is an iron(II)-dependent dioxygenase involved in the aerobic degradation of 5-nitroanthranilic acid by the bacterium Bradyrhizobium sp. It catalyzes the opening of the 5-nitrosalicylate aromatic ring, a key step in the degradation pathway. Besides 5-nitrosalicylate, the enzyme is also active towards 5-chlorosalicylate.

Indicator-driven data calibration of expert interviews in a configurational study.

Expert interviews can provide interesting data for the use in qualitative comparative analysis (QCA) to investigate complex social phenomena. To guide the challenging task of data calibration from qualitative data sets, techniques have already been suggested for the transformation of qualitative data into fuzzy sets. The current article follows existing guidelines and extends them with a system for indicator-based data calibration of expert interviews.

Comparative Analysis of the Conversion of Mandelonitrile and 2-Phenylpropionitrile by a Large Set of Variants Generated from a Nitrilase Originating from EBC191.

The arylacetonitrilase from the bacterium EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts of the corresponding amides. The enzyme has been analysed by site-specific mutagenesis and more than 50 different variants have been generated and analysed for the conversion of (,)-mandelonitrile and (,)-2-phenylpropionitrile.

Conversion of phenylglycinonitrile by recombinant Escherichia coli cells synthesizing variants of the arylacetonitrilase from Pseudomonas fluorescens EBC191.

The conversion of phenylglycinonitrile (2-aminophenylacetonitrile) by Escherichia coli strains was studied, which recombinantly expressed the arylacetonitrilase (NitA) from Pseudomonas fluorescens EBC191 and different nitrilase variants with altered reaction specificities. The whole-cell catalysts which formed the wild-type nitrilase converted (R,S)-phenylglycinonitrile preferentially to (S)-phenylglycine with a low degree of enantioselectivity. A recombinant strain which formed a variant of NitA produced mainly (S)-phenylglycine amide from (R,S)-phenylglycinonitrile and a second variant showed an almost complete enantioconversion and produced (R)-phenylglycine and left (S)-phenylglycinonitrile.

Substrate promiscuity and active site differences in gentisate 1,2-dioxygenases: electron paramagnetic resonance study.

Gentisate 1,2-dioxygenases (GDOs) are non-heme iron enzymes that catalyze the oxidation of dihydroxylated aromatic substrate, gentisate (2,5-dihydroxybenzoate). Salicylate 1,2-dioxygenase (SDO), a member of the GDO family, performs the ring scission of monohydroxylated substrates such as salicylate, thereby oxidizing a broader range of substrates compared to GDOs. Although the two types of enzymes share a high degree of sequence similarity, the origin of substrate specificity between SDO and GDOs is not understood.

Conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191.

The conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 (NitA) was analyzed. The nitrilase hydrolysed a wide range of aliphatic mono- and dinitriles and showed a preference for unsaturated aliphatic substrates containing 5-6 carbon atoms. In addition, increased reaction rates were also found for aliphatic nitriles carrying electron withdrawing substituents (e.

Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates.

The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C.

Spontaneous release of fluoride during the dioxygenolytic cleavage of 5-fluorosalicylate by the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans BN12.

The alpha-Proteobacterium Pseudaminobacter salicylatoxidans BN12 forms a peculiar gentisate 1,2-dioxygenase (SDO) that oxidatively cleaves gentisate (2,5-dihydroxybenzoate) and additionally 1-hydroxy-2-naphthoate, salicylate and various amino-, chloro-, fluoro-, hydroxy- and methylsalicylates. In the present study, the conversion of 5-fluorosalicylate by this enzyme was analysed using various analytical techniques. Spectrophotometric assays showed that the conversion of 5-fluorosalicylate by the purified enzyme resulted in the formation of a new unstable intermediate showing an absorbance maximum at λmax = 292 nm.

Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans.

The genome of the α-proteobacterium Pseudaminobacter salicylatoxidans codes for a ferrous iron containing ring-fission dioxygenase which catalyzes the 1,2-cleavage of (substituted) salicylate(s), gentisate (2,5-dihydroxybenzoate), and 1-hydroxy-2-naphthoate. Sequence alignments suggested that the "salicylate 1,2-dioxygenase" (SDO) from this strain is homologous to gentisate 1,2-dioxygenases found in bacteria, archaea and fungi. In the present study the catalytic mechanism of the SDO and gentisate 1,2-dioxygenases in general was analyzed based on sequence alignments, mutational and previously performed crystallographic studies and mechanistic comparisons with "extradiol- dioxygenases" which cleave aromatic nuclei in the 2,3-position.

Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5-SMG7 and SMG6.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes mRNAs with premature stop codons and targets them for rapid degradation. Evidence from previous studies has converged on UPF1 as the central NMD factor. In human cells, the SMG1 kinase phosphorylates UPF1 at the N-terminal and C-terminal tails, in turn allowing the recruitment of the NMD factors SMG5, SMG6 and SMG7.

A single TCR alpha-chain with dominant peptide recognition in the allorestricted HER2/neu-specific T cell repertoire.

T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2(369)) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells.

Formin-like 1 (FMNL1) is regulated by N-terminal myristoylation and induces polarized membrane blebbing.

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1gamma) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD).

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies.

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected.

Effect of novel modulators of protein kinase C activity upon chemotherapy-induced differentiation and apoptosis in myeloid leukemic cells.

Modulation of protein kinase C (PKC) activity has been demonstrated to either prevent or enhance drug-induced apoptosis in various tissue types. We tested four novel modulators of PKC activity in comparison to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for the capability to affect differentiation, cell cycle progression and apoptosis in the human myeloid leukemia cell lines U937 and HL-60. Farnesyl thiotriazole (FTT) and N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) are both direct activators of PKC, whereas 6-(2-(4-[(4-fluorophe-nyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quin-azolinone (R59949) are diacyl glycerol kinase inhibitors that activate PKC by enhancing the levels of the endogenous ligand diacyl glycerol.