Resorcinol Hydroxylase of Azoarcus anaerobius: Molybdenum Dependence, Activity, and Heterologous Expression.

The obligately anaerobic, denitrifying bacterium Azoarcus anaerobius strain LuFRes1 grows with resorcinol (1,3-dihydroxybenzene) as sole carbon and energy source. Resorcinol is oxidized to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by resorcinol hydroxylase (RH), an inducible membrane-bound enzyme. Sequence comparison places resorcinol hydroxylase into the group of anaerobic molybdopterin oxidoreductases and dimethyl sulfoxide reductase-like enzymes. In the large subunit, a molybdopterin-binding domain was predicted, and the small subunit most likely contains two [4Fe-4S] centers. Growth of molybdate-starved cells was inhibited by tungstate, and in vitro resorcinol hydroxylase activity was inhibited by arsenite and selenite that are known to inhibit molybdenum-containing enzymes. The two genes encoding resorcinol hydroxylase could be expressed in Escherichia coli but the products remained in inclusion bodies. All attempts to purify RH from A. anaerobius or to produce soluble, active RH in E. coli failed. Nevertheless, RH was produced as a C-terminally Strep-tagged protein from plasmid pSKM1 in Thauera aromatica AR1 transconjugants carrying a transposon insertion in the coding gene for the large (ΔrhL) or the small subunit (ΔrhS) of RH from cosmid R+. RH in the membrane fraction of wild-type transconjugant T. aromatica AR1/R+ showed a specific activity of 80 mU mg-1, and the specific activity of RH in the membranes of the complemented mutants was in the same range (80-95 mU mg-1). We conclude that RH of A. anaerobius is a membrane-bound molybdoenzyme consisting of two subunits which might require a further loosely bound subunit as membrane anchor.

Structural insights into the binding of vascular endothelial growth factor-B by VEGFR-1(D2): recognition and specificity.

The formation of blood vessels (angiogenesis) is a highly orchestrated sequence of events involving crucial receptor-ligand interactions. Angiogenesis is critical for physiological processes such as development, wound healing, reproduction, tissue regeneration, and remodeling. It also plays a major role in sustaining tumor progression and chronic inflammation. Vascular endothelial growth factor (VEGF)-B, a member of the VEGF family of angiogenic growth factors, effects blood vessel formation by binding to a tyrosine kinase receptor, VEGFR-1. There is growing evidence of the important role played by VEGF-B in physiological and pathological vasculogenesis. Development of VEGF-B antagonists, which inhibit the interaction of this molecule with its cognate receptor, would be important for the treatment of pathologies associated specifically with this growth factor. In this study, we present the crystal structure of the complex of VEGF-B with domain 2 of VEGFR-1 at 2.7 A resolution. Our analysis reveals that each molecule of the ligand engages two receptor molecules using two symmetrical binding sites. Based on these interactions, we identify the receptor-binding determinants on VEGF-B and shed light on the differences in specificity towards VEGFR-1 among the different VEGF homologs.

Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: enhanced T cell stimulatory capacity despite IDO.

The exclusive ability of dendritic cells (DCs) to stimulate primary and secondary immune responses favors the use of antigen-loaded human monocyte-derived DCs (MoDCs) in vaccinations against tumors. Previous studies demonstrated that PGE(2) is fundamental during MoDC maturation to facilitate migration toward lymph node-derived chemokines. A recent study challenged the use of PGE(2), as PGE(2) induced IDO in mature MoDCs. In MoDCs compatible for clinical use, we now demonstrate that PGE(2) is responsible for IDO induction if matured by soluble CD40 ligand, LPS, or cytokines. In contrast, IDO expression in MoDCs matured by TLR3 triggering occurs independently of PGE(2). It is surprising that despite active IDO protein, MoDCs matured with PGE(2) display a greater potential to stimulate naïve CD4(+) and CD8(+) T cell proliferation, which is not increased further by IDO inhibition. Moreover, we found elevated levels of tryptophanyl-tRNA-synthetase (TTS) in T cells cultured with PGE(2)-matured MoDCs. Our data demonstrate that PGE(2) induces IDO in MoDCs but that T cell-stimulating capacities of PGE(2)-matured MoDCs overcome IDO activity, probably through TTS induction. As PGE(2) is critical for DC migration and enhances the capability of MoDCs to induce T cell proliferation, we highly recommend supplementing DC maturation stimuli with PGE(2) for use in clinical trials.

Heterologous expression and identification of the genes involved in anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) in Azoarcus anaerobius.

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R(+), carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R(+) carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R(+), eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.