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The Fe(II)- and [alpha]-ketoglutarate ([alpha]KG)-dependent dioxygenases use mononuclear nonheme iron centers to effect hydroxylation of their substrates and decarboxylation of their cosubstrate, [alpha]KG, to C[O.sub.2] and succinate. Our recent dissection of the mechanism of taurine:[alpha]KG dioxygenase (TauD), a member of this enzyme family, revealed that two transient complexes accumulate during catalysis in the presence of saturating substrates. The first complex contains the long-postulated C-H-cleaving Fe(IV)-oxo intermediate, J, and the second is an enzyme product(s) complex. Here, we demonstrate the accumulation of two transient complexes in the reaction of a prolyl-4-hydroxylase (P4H), a functional homologue of human [alpha]KG-dependent dioxygenases with essential roles in collagen biosynthesis and oxygen sensing. The kinetic and spectroscopic properties of these two P4H complexes suggest that they are homologues of the TauD intermediates. Most notably, the first exhibits optical absorption and Mossbauer spectra similar to those of J and, like J, a large substrate deuterium kinetic isotope on its decay. The close correspondence of the accumulating states in the P4H and TauD reactions supports the hypothesis of a conserved mechanism for substrate hydroxylation by enzymes in this family. Fe(IV)-oxo intermediates | hydroxylation | nonheme iron enzymes | oxygen activation

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 I[kappa]B 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 I[kappa]B[alpha] 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 ARD-containing 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-[kappa]B | 2-oxoglutarate-dependent dioxygenase | protein hydroxylation

Electrolytic systems containing a platinum electrode and a redox mediator with purified recombinant proteins containing P450A1 are applicable to various P450s for the synthesis of stereo and regio-specific hydroxylated products. Experimental results show that NADPH can be replaced by these systems for the electrocatalytically mediated omega-hydroxylation of lauric acid.

Synthesis of Tyr in the human body occurs by hydroxylation of the indispensable amino acid Phe. Until now, it was believed that in humans, this process was restricted to the liver, but we provide compelling evidence of production of Tyr from Phe in the kidney. To determine whether the human kidney produces Tyr, we measured Tyr balance, the Tyr appearance rate, and the Phe-to-Tyr conversion in 12 healthy human subjects by using [[sup.15]N]Phe and [[sup.2][H.sub.4]]Tyr as tracers. Renal plasma flow was measured by using paraaminohippurate, and sampling from the femoral artery and renal veins was performed. The results were compared with those obtained in 12 control subjects undergoing hepatic vein catheterization and infusion of identical tracers. In all 12 subjects, there was a net uptake of Phe by the kidney (2.2 [+ or -] 1.2 [micro]mol/min), whereas Tyr was released (5.3 [+ or -] 1.5 [micro]mol/min). In contrast, there was a net uptake of both Phe (9.5 [+ or -] 1.2 [micro]mol/min) and Tyr (14.3 [+ or -] 1.3 [micro]mol/min) by the splanchnic bed. Phe conversion to Tyr occurred at a rate of 5.2 [+ or -] 1.2 [micro]mol/min in kidney and 3.0 [+ or -] 0.7 [micro]mol/min in the splanchnic bed. The kidney contributed a substantial amount of Tyr to the systemic circulation where the splanchnic bed was a net remover of Tyr. Our results demonstrate that the kidney is the major donor of Tyr to the systemic circulation by its conversion of Phe to Tyr. This observation may have important clinical implications for patients with both renal and hepatic disease, who may be at riskof Phe overloading and Tyr deficiency, and it should be considered when parenteral or enteral nutrients are administered rich in Phe and low in Tyr.