Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues. Hydroxylation of specific prolyl residues by 2-oxoglutarate (2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-␣. Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-␣ abrogates interaction with p300, preventing transcriptional activation. Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-␣. Since FIH contains certain motifs present in iron-and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase. Assays using recombinant FIH and HIF-␣ fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen. Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.Hypoxia in animals activates a broad range of homeostatic responses via induction of a transcriptional complex termed hypoxia-inducible factor (HIF) 1 (1, 2). HIF is a heterodimer of ␣-and ␤-subunits with regulation by dioxygen availability being mediated by post-translational modification of the ␣-subunits (1, 2). In mammalian cells, two HIF-␣ subunit isoforms (HIF-1␣ and HIF-2␣) are regulated by dioxygen levels. Each HIF-␣ protein contains an internal oxygen-dependent degradation domain possessing targeting motifs for proteolytic regulation and a C-terminal transactivation domain (CAD) independently regulated by dioxygen, irrespective of changes in protein abundance, through interaction with the CH1 domain of the co-activator p300 (for review, see Ref.3).The oxygen-dependent degradation of HIF-␣ by proteolysis is regulated by the hydroxylation of specific prolyl residues (Pro-402 and Pro-564 in human HIF-1␣) that mediate recognition of HIF-␣ by the von Hippel-Lindau (VHL) ubiquitinylation complex and consequent proteasomal destruction (4 -7). Combined structural analysis and genetic approaches led to the identification of three isoforms of human HIF prolyl hydroxylase (PHD1-3, prolyl hydroxylase domain) together with homologues in a range of organisms (8,9). In vitro analyses together with sequence and mutational analyses identified these as belonging to a subfamily of the Fe(II)-and 2-oxoglutarate (2-OG)-dependent oxygenases (4,5,8,9). Limiting oxygen availability in hypoxia, or direct inhibition of the PHD enzymes by cobaltous ions and iron chelators, allows HIF-␣ to escape hydroxylation and recognition by pVHL, providing insights into the mechanism by which these stimuli suppress HIF-␣ degradation and activate the transcriptional casca...

Flavonoids are common colorants in plants and have long-established biomedicinal properties. Anthocyanidin synthase (ANS), a 2-oxoglutarate iron-dependent oxygenase, catalyzes the penultimate step in the biosynthesis of the anthocyanin class of flavonoids. The crystal structure of ANS reveals a multicomponent active site containing metal, cosubstrate, and two molecules of a substrate analog (dihydroquercetin). An additional structure obtained after 30 min exposure to dioxygen is consistent with the oxidation of the dihydroquercetin to quercetin and the concomitant decarboxylation of 2-oxoglutarate to succinate. Together with in vitro studies, the crystal structures suggest a mechanism for ANS-catalyzed anthocyanidin formation from the natural leucoanthocyanidin substrates involving stereoselective C-3 hydroxylation. The structure of ANS provides a template for the ubiquitous family of plant nonhaem oxygenases for future engineering and inhibition studies.

Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3␤-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate-and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate.

Niemann-Pick disease type C (NP-C) is a neurovisceral lysosomal cholesterol trafficking and lipid storage disorder caused by mutations in one of the two genes, NPC1 or NPC2. Diagnosis has often been a difficult task, due to the wide range in age of onset of NP-C and clinical presentation of the disease, combined with the complexity of the cell biology (filipin) laboratory testing, even in combination with genetic testing. This has led to substantial delays in diagnosis, largely depending on the access to specialist centres and the level of knowledge about NP-C of the physician in the area. In recent years, advances in mass spectrometry has allowed identification of several sensitive plasma biomarkers elevated in NP-C (e.g. cholestane-3β,5α,6β-triol, lysosphingomyelin isoforms and bile acid metabolites), which, together with the concomitant progress in molecular genetic technology, have greatly impacted the strategy of laboratory testing. Specificity of the biomarkers is currently under investigation and other pathologies are being found to also result in elevations. Molecular genetic testing also has its limitations, notably with unidentified mutations and the classification of new variants. This review is intended to increase awareness on the currently available approaches to laboratory diagnosis of NP-C, to provide an up to date, comprehensive and critical evaluation of the various techniques (cell biology, biochemical biomarkers and molecular genetics), and to briefly discuss ongoing/future developments. The use of current tests in proper combination enables a rapid and correct diagnosis in a large majority of cases. However, even with recent progress, definitive diagnosis remains challenging in some patients, for whom combined genetic/biochemical/cytochemical markers do not provide a clear answer. Expertise and reference laboratories thus remain essential, and further work is still required to fulfill unmet needs.

The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O 2 free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O 2 migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k cat /K m (O 2 ), supporting multiple dynamic pathways for molecular oxygen to reach the active site.Copper amine oxidases are ubiquitous copper containing enzymes that oxidize primary amines to aldehydes through the reduction of molecular oxygen to hydrogen peroxide. CAO 2 catalysis is dependent upon the protein-derived cofactor 2,4,5-trihydroxyphenylalaninequinone (TPQ). The TPQ is derived from an endogenous tyrosine through a self-catalytic process requiring only molecular oxygen and Cu(II) (see Fig. 1a) (1).Hansenula polymorpha 3 amine oxidase is the eukaryotic CAO that has been kinetically characterized in the most detail (2-7). HPAO follows a Bi Bi ping-pong reaction mechanism that can be expressed as two half-reactions, reductive and oxidative (see Fig. 1b). In the reductive half-reaction the enzyme oxidizes a primary amine to an aldehyde, generating the 2e Ϫ reduced aminoquinol form of the cofactor. In the subsequent oxidative half-reaction molecular oxygen is reduced to hydrogen peroxide via cofactor reoxidation to TPQ. Biochemical studies from several different laboratories have led to mechanistic proposals for the catalytic cycle of CAOs (8, 9). These studies have given significant insight into the mechanism for the reductive half-reaction (10). However, the details surrounding the activation of molecular oxygen both in terms of the biogenesis of the TPQ (11, 12) and of the catalytic oxidative half-reaction, remain the subject of intense study (13-16). The utilization of copper as a redox center has been the focus of recent controversy. Because CAOs contain a copper ion in their active site, chemical intuition suggests Cu(I) as the O 2 -activating species to give Cu(II)-superoxide (17). Upon a...

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.The integrity of the genome is maintained by a set of DNA repair enzymes, including those that repair alkylation damage (1). DNA can be alkylated by a variety of agents that occur both exogenously and endogenously (2, 3). Alkylating agents are used in some chemotherapy treatments, and alkylated DNA bases have been detected in the urine of smokers (4). AlkB catalyzes demethylation of DNA and along with AlkA, AidB, and Ada is one of four proteins involved in the adaptive response to alkylation damaged DNA in Escherichia coli (5). Ada, which has been termed the "suicidal repair protein," contains two active sites, one of which demethylates O 6 -methylguanine by irreversible transfer of the methyl group to a cysteine (5, 6).The second active site removes methyl groups from S-methyl phosphoesters by nucleophilic methylation of another cysteine residue (5,7,8). Methylation of the latter cysteine converts Ada into a strong transcriptional activator of both its own production and the other three proteins of the adaptive response (9). AlkA is a DNA glycosylase with a wide substrate selectivity, including excision of cytotoxic 3-methyladenine residues (10). AlkA "flips" the damaged base out of the DNA double helix by inserting Leu-125 into the position occupied by the damaged base (11)...

Niemann–Pick disease type C (NP-C) is a rare, autosomal-recessive, progressive neurological disease caused by mutations in either the NPC1 gene (in 95% of cases) or the NPC2 gene. This observational, multicentre genetic screening study evaluated the frequency and phenotypes of NP-C in consecutive adult patients with neurological and psychiatric symptoms. Diagnostic testing for NP-C involved NPC1 and NPC2 exonic gene sequencing and gene dosage analysis. When available, results of filipin staining, plasma cholestane-3β,5α,6β-triol assays and measurements of relevant sphingolipids were also collected. NPC1 and NPC2 gene sequencing was completed in 250/256 patients from 30 psychiatric and neurological reference centres across the EU and USA [median (range) age 38 (18–90) years]. Three patients had a confirmed diagnosis of NP-C; two based on gene sequencing alone (two known causal disease alleles) and one based on gene sequencing and positive filipin staining. A further 12 patients displayed either single mutant NP-C alleles (8 with NPC1 mutations and 3 with NPC2 mutations) or a known causal disease mutation and an unclassified NPC1 allele variant (1 patient). Notably, high plasma cholestane-3β,5α,6β-triol levels were observed for all NP-C cases (n = 3). Overall, the frequency of NP-C patients in this study [1.2% (95% CI; 0.3%, 3.5%)] suggests that there may be an underdiagnosed pool of NP-C patients among adults who share common neurological and psychiatric symptoms.

Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2–50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.