Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor

The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35A) and of Notch peptides bound to FIH (2.4-2.6A) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.     

The selectivity and inhibition of AlkB

AlkB is one of four proteins involved in the adaptive response to DNA alkylation damage in Escherichia coli and is highly conserved from bacteria to humans. Recent analyses have verified the prediction that AlkB is a member of the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family of enzymes. AlkB mediates repair of methylated DNA by direct demethylation of 1-methyladenine and 3-methylcytosine lesions. Other members of the Fe(II) and 2OG-dependent oxygenase family, including those involved in the hypoxic response, are targets for therapeutic intervention. Assays measuring 2OG turnover were used to investigate the selectivity of AlkB. 1-Methyladenosine, 1-methyl-2'-deoxyadenosine, 3-methylcytidine, and 3-methyl-2'-deoxycytidine all stimulated 2OG turnover by AlkB but were not demethylated indicating an uncoupling of 2OG and prime substrate oxidation and that oligomeric DNA is required for hydroxylation and subsequent demethylation. In contrast the equivalent unmethylated nucleosides did not stimulate 2OG turnover indicating that the presence of a methyl group in the substrate is important in initiating oxidation of 2OG. Stimulation of 2OG turnover by 1-methyladenosine was highly dependent on the presence of a reducing agent, ascorbate or dithiothreitol. Following the observation that AlkB is inhibited by high concentrations of 2OG, analogues of 2OG, including 2-mercaptoglutarate, were found to specifically inhibit AlkB. The flavonoid quercetin inhibits both AlkB and the 2OG oxygenase factor-inhibiting hypoxia-inducible factor (FIH) in vitro. FIH inhibition by quercetin occurs in the presence of excess iron indicating a specific interaction, while the inhibition of AlkB by quercetin is, predominantly, due to nonspecific iron chelation.     

Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay

The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent K(m) (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent K(m) (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1alpha. The present results, albeit obtained under non-physiological conditions, imply that the apparent K(m) (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.     

Asparaginyl β-Hydroxylation of Proteins Containing Ankyrin Repeat Domains Influences Their Stability and Function

Recent reports have provided evidence that the β-hydroxylation of conserved asparaginyl residues in ankyrin repeat domain (ARD) proteins is a common posttranslational modification in animal cells. Here, nuclear magnetic resonance (NMR) and other biophysical techniques are used to study the effect of asparaginyl β-hydroxylation on the structure and stability of ‘consensus’ ARD proteins. The NMR analyses support previous work suggesting that a single β-hydroxylation of asparagine can stabilize the stereotypical ARD fold. A second asparaginyl β-hydroxylation causes further stabilization. In combination with mutation studies, the biophysical analyses reveal that the stabilizing effect of β-hydroxylation is, in part, mediated by a hydrogen bond between the asparaginyl β-hydroxyl group and the side chain of a conserved aspartyl residue, two residues to the N-terminal side of the target asparagine. Removal of this hydrogen bond resulted in reduced stabilization by hydroxylation. Formation of the same hydrogen bond is also shown to be a factor in inhibiting binding of hydroxylated ARDs to factor-inhibiting hypoxia-inducible factor (FIH). The effects of hydroxylation appear to be predominantly localized to the target asparagine and proximal residues, at least in the consensus ARD protein. The results reveal that thermodynamic stability is a factor in determining whether a particular ARD protein is an FIH substrate; a consensus ARD protein with three ankyrin repeats is an FIH substrate, while more stable consensus ARD proteins, with four or five ankyrin repeats, are not. However, NMR studies reveal that the consensus protein with four ankyrin repeats is still able to bind to FIH, suggesting that FIH may interact in cells with natural ankyrin repeats without resulting hydroxylation. Overall, the work provides novel biophysical insights into the mechanism by which asparaginyl β-hydroxylation stabilizes the ARD proteins and reduces their binding to FIH.     

Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases

Mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases comprise a large family of enzymes that utilize an Fe(IV)-oxo intermediate to initiate diverse oxidative transformations with important biological roles. Here, four of the major types of Fe(II)/2OG-dependent reactions are detailed: hydroxylation, halogenation, ring formation, and desaturation. In addition, an atypical epimerization reaction is described. Studies identifying several key intermediates in catalysis are concisely summarized, and the proposed mechanisms are explained. In addition, a variety of other transformations catalyzed by selected family members are briefly described to further highlight the chemical versatility of these enzymes.     

Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)

The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.     

Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases

The post-translational hydroxylation of prolyl and lysyl residues, as catalyzed by 2-oxoglutarate (2OG)-dependent oxygenases, was first identified in collagen biosynthesis. 2OG oxygenases also catalyze prolyl and asparaginyl hydroxylation of the hypoxia-inducible factors that play important roles in the adaptive response to hypoxia. Subsequently, they have been shown to catalyze N-demethylation (via hydroxylation) of N^ϵ-methylated histone lysyl residues, as well as hydroxylation of multiple other residues. Recent work has identified roles for 2OG oxygenases in the modification of translation-associated proteins, which in some cases appears to be conserved from microorganisms through to humans. Here we give an overview of protein hydroxylation catalyzed by 2OG oxygenases, focusing on recent discoveries.