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...

Diverse insect species harbor symbiotic bacteria, which play important roles such as provisioning nutrients and providing defense against natural enemies [1-6]. Whereas nutritional symbioses are often indispensable for both partners, defensive symbioses tend to be of a facultative nature [1-12]. The Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits Liberibacter spp. (Alphaproteobacteria), causing the devastating citrus greening disease or Huanglongbing [13, 14]. In a symbiotic organ called the bacteriome, D. citri harbors two distinct intracellular symbionts: a putative nutrition provider, Carsonella_DC (Gammaproteobacteria), and an unnamed betaproteobacterium with unknown function [15], for which we propose the name "Candidatus Profftella armatura." Here we report that Profftella is a defensive symbiont presumably of an obligate nature with an extremely streamlined genome. The genomes of Profftella and Carsonella_DC were drastically reduced to 464,857 bp and 174,014 bp, respectively, suggesting their ancient and mutually indispensible association with the host. Strikingly, 15% of the small Profftella genome encoded horizontally acquired genes for synthesizing a novel polyketide toxin. The toxin was extracted, pharmacologically and structurally characterized, and designated diaphorin. The presence of Profftella and its diaphorin-biosynthetic genes was perfectly conserved in the world's D. citri populations.

Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-␣ subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Å resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded ␤-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.erythropoietin ͉ dioxygenase ͉ hypoxic response ͉ 2-oxoglutarate I n metazoans the ␣͞␤ heterodimeric hypoxia-inducible transcription factor (HIF) (1) regulates the transcription of an array of genes including those coding for glycolytic enzymes, erythropoietin, and VEGF. The levels and transcriptional activity of the HIF-␣, but not the HIF-␤, subunit are regulated by oxygen. Hydroxylation of either Pro-402 or Pro-564 in human HIF-1␣ (2, 3) within the C-terminal oxygen-dependent degradation domain (CODDD) enables its binding to the von Hippel-Lindau protein (pVHL), a targeting element of the E3-ubiquitin ligase; subsequent ubiquitylation leads to proteasomal degradation of HIF-␣ (for reviews, see refs. 4 -7). In humans, this mechanism is augmented by hydroxylation of an asparagine residue in the C-terminal transcriptional activation domain (8); this modification blocks interaction of HIF-1␣ with the CBP͞p300 coactivator, thereby disabling HIFmediated transcription.Hydroxylation of HIF-1␣ is catalyzed by four 2-oxoglutarate (2OG) dioxygenases: three prolyl hydroxlyases (PHD 1, 2, and 3) (also known as HPH 3, 2, and 1 and EGLN 2, 1, and 3; refs. 9-11) and an asparaginyl hydroxylase [factor inhibiting HIF (FIH); refs. 12 and 13]. The available evidence implicates PHD2 as the most important HIF hydroxylase in down-regulating the hypoxic response during normoxia (5,7,14,15).The HIF hydroxylases are Fe(II) and 2OG-dependent dioxygenases (16, 17); their requirement for dioxygen has led to their characterization as cellular oxygen sensors (refs. 9 -11, 18, and 19; Fig. 1a). The first 2OG dioxygenase to be identified was procollagen prolyl-hydroxylase, which like the PHDs catalyzes trans-4-hydroxylation reactions. Pro...

It is held as a paradigm that ribosomally synthesized peptides and proteins contain only l-amino acids. We demonstrate a ribosomal origin of the marine sponge-derived polytheonamides, exceptionally potent, giant natural-product toxins. Isolation of the biosynthetic genes from the sponge metagenome revealed a bacterial gene architecture. Only six candidate enzymes were identified for 48 posttranslational modifications, including 18 epimerizations and 17 methylations of nonactivated carbon centers. Three enzymes were functionally validated, which showed that a radical S-adenosylmethionine enzyme is responsible for the unidirectional epimerization of multiple and different amino acids. Collectively, these complex alterations create toxins that function as unimolecular minimalistic ion channels with near-femtomolar activity. This study broadens the biosynthetic scope of ribosomal systems and creates new opportunities for peptide and protein bioengineering.

Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the IB family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and IB␣ were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARDcontaining proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.NF-B ͉ 2-oxoglutarate-dependent dioxygenase ͉ protein hydroxylation C ells react to variation in oxygen availability with adaptive responses that involve changes in most basic cellular functions. Analysis of the transcriptional component of this response has defined pathways that regulate hypoxia-inducible factor (HIF) by posttranslational hydroxylation of specific residues. HIF is an ␣͞␤ heterodimer that binds hypoxia response elements in a range of hypoxia-inducible genes (for review, see ref. 1). Regulation is mediated by the ␣-subunits and involves dual mechanisms controlling both the abundance and activity of the protein. Thus, hydroxylation of specific prolyl residues promotes interaction with the von Hippel-Lindau E3 ligase and hence proteolysis, whereas hydroxylation of a C-terminal Asn residue blocks recruitment of the coactivators p300͞CBP. The prolyl and asparaginyl hydroxylase enzymes that catalyze these reactions are 2-oxoglutarate (2-OG) and Fe(II)-dependent dioxygenases that couple the oxidative decarboxylation of 2-OG with oxidation of peptidyl substrates. Dioxygen is an obligate cosubstrate, and reductions in the rate of hydroxylation during hypoxia allow HIF-␣ to escape VHLmediated destruction and to activate transcription (for reviews, see refs. 2 and 3).HIF prolyl hydroxylation is catalyzed by three enzymes, PHD1, -2, and -3 (equivalent to EGLN2, -1, and -3 and HPH-3, -2, and -1). HIF Asn hydroxylation is catalyzed by a more distantly related 2-OG-dependent dioxygenase, factor inhibiting HIF (FIH) (for reviews, see refs. 2 and 3). A key question raised by these findings is whether the roles of all four dioxygenases are specific to HIF regulation, or whether one or more have alternative substrates. Several studies have identified proteins that interact to modulate HIF hydroxylase activity (4) or ...