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The Escherichia coli DNA repair enzyme AlkB is a 2-oxoglutarate (2OG)-dependent Fe2+ binding dioxygenase that removes methyl lesions from DNA and RNA. To date, nine human AlkB homologues are known: ABH1 to ABH8 and the obesity-related FTO. Similar to AlkB, these homologues exert their activity on nucleic acids, although for some homologues the biological substrate remains to be identified. 2OG dioxygenases require binding of the cofactors Fe2+ and 2OG in the active site to form a catalytically competent complex. We present a structural analysis of AlkB using NMR, fluorescence, and CD spectroscopy to show that AlkB is a dynamic protein exhibiting different folding states. In the absence of the cofactors Fe2+ and 2OG, apoAlkB is a highly dynamic protein. Binding of either Fe2+ or 2OG alone does not significantly affect the protein dynamics. Formation of a fully folded and catalytically competent holoAlkB complex only occurs when both 2OG and Fe2+ are bound. These findings provide the first insights into protein folding of 2OG-dependent dioxygenases. A role for protein dynamics in the incorporation of the metal cofactor is discussed. [ABSTRACT FROM AUTHOR]

The membrane-bound [NiFe]-hydrogenase fromAllochromatium vinosumcan occur in several inactive or active states. This study presents the first systematic infrared characterisation of theA. vinosumenzyme, with emphasis on the spectro-electrochemical properties of the inactive/active transition. This transition involves an energy barrier, which can be overcome at elevated temperatures. The reduced Ready enzyme can exist in two different inactive states, which are in an apparent acid-base equilibrium. It is proposed that a hydroxyl ligand in a bridging position in the Ni-Fe site is protonated and that the formed water molecule is subsequently removed. This enables the active site to bind hydrogen in a bridging position, allowing the formation of the fully active state of the enzyme. It is further shown that the active site in enzyme reduced by 1 bar H2 can occur in three different electron paramagnetic resonance (EPR)-silent states with a different degree of protonation. [ABSTRACT FROM AUTHOR]

The soluble, cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-NAD+ reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity – the reduction of NAD+ by H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 was not dependent on the presence of FMN-a. Enzyme devoid of FMN-a could not be activated by NADH. As NADH-dehydrogenase activity was not dependent on the presence of FMN-a, and because FMN-b did not dissociate from the reduced enzyme, we conclude that FMN-b is functional in the NADH-dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN-a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed. [ABSTRACT FROM AUTHOR]

Cancer therapy frequently fails due to the emergence of resistance. Many tumors include phenotypically immature tumor cells, which have been implicated in therapy resistance. Neuroblastoma cells can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) state. They differ in epigenetic landscape and transcription factors, and MES cells are more resistant to chemotherapy. Here we analyzed the response of MES cells to targeted drugs. Activating anaplastic lymphoma kinase ( ALK ) mutations are frequently found in neuroblastoma and ALK inhibitors (ALKi) are in clinical trials. ALKi treatment of ADRN neuroblastoma cells with a tumor-driving ALK mutation induced cell death. Conversely, MES cells did not express either mutant or wild-type ALK and were resistant to ALKi, and MES cells formed tumors that progressed under ALKi therapy. In assessing the role of MES cells in relapse development, TRAIL was identified to specifically induce apoptosis in MES cells and to suppress MES tumor growth. Addition of TRAIL to ALKi treatment of neuroblastoma xenografts delayed relapses in a subset of the animals, suggesting a role for MES cells in relapse formation. While ADRN cells resembled normal embryonal neuroblasts, MES cells resembled immature precursor cells, which also lacked ALK expression. Resistance to targeted drugs can therefore be an intrinsic property of immature cancer cells based on their resemblance to developmental precursors. SIGNIFICANCE: In neuroblastoma, mesenchymal tumor cells lack expression of the tumor-driving ALK oncogene and are resistant to ALKi, but dual treatment with ALKi and mesenchymal cell-targeting TRAIL delays tumor relapse., (©2021 American Association for Cancer Research.)