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An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD iso-form preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of 'paired' enzyme-substrate reporters to evaluate the potency of 'branched tail' oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform. [ABSTRACT FROM AUTHOR]

Adaptaquin, 7-{(4-chlorophenyl)[(3-hydroxypyridin-2-yl)amino]methyl}quinolin-8-ol (1), was identified in our earlier study as a hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor by high-throughput screening of cell lines, which stably express the reporter construct encoding a fusion protein composed of the HIF oxygen degradable domain and firefly luciferase (HIF1 ODD-luc). Adaptaquin is a specific inhibitor of this enzyme and exhibits neuroprotective properties in cell models of oxidative stress and in vivo models of hemorrhagic stroke. The role of the chiral carbon atom in the inhibitor molecule was studied in order to further improve the adaptaquin structure. The separation into two enantiomers was performed. Both enantiomers were shown to equally activate the HIF1 ODD-luc reporter. The adaptaquin structure can be significantly modified and made more sophisticated in order to improve specificity toward this group of enzymes and subsequently toward individual isozymes. [ABSTRACT FROM AUTHOR]

The flavin adenine dinucleotide-containing domain of the protein MICAL (molecule interacting with CasL) is a monooxygenase. This protein plays an important role in the regulation of axonal guidance. However, the mechanism of this process has been unknown until recently. Only two years ago, F-actin was found to be the physiological substrate. The oxidation of methionine residues of this substrate is catalyzed by MICAL, resulting in the depolymerization of actin. A year ago it was shown that methionine sulfoxide reductase (MSR) catalyzes the reverse reaction of reduction of oxidized actin methionines. Therefore, this couple of proteins is currently a target for manipulation of the ability of neurons to regenerate their axons. The present study deals with different approaches to the regulation of the activity of MICAL and MSR and the design of inhibitors of the former enzyme. [ABSTRACT FROM AUTHOR]

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely β2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed. [ABSTRACT FROM AUTHOR]

The recombinant horseradish peroxidase and its single-point F41H mutant have been reactivated from E. coli inclusion bodies. The influence of the mutation on the heme entrapment, stability and activity of the enzyme was demonstrated. The catalytic rate constants for HO cleavage and ammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the elimination of the ABTS oxidation product is not a rate-determining step for the mutant. The F41H replacement results in significant changes of kinetics of iodide ion oxidation. The reaction rate is linear to the concentrations of iodide, HO, and the enzyme. The results suggest the direct interaction of iodide with the porphyrin ring of the heme. The decrease in ABTS oxidation activity accompanied by retention of activity in iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iodide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved. [ABSTRACT FROM AUTHOR]