Hydroxylase Reaction Research Articles

7-Substituted pterins: Formation during phenylalanine hydroxylation in the absence of dehydratase

Previously we described a new form of human hyperphenylalaninemia characterized by the formation of 7-substituted pterins. We present evidence strongly suggesting that the 7-substituted pterins are formed by rearrangement of 6-substituted pterins. This rearrangement occurs during the phenylalanine hydroxylase reaction cycle which normally involves the enzymes phenylalanine hydroxylase, pterin-4a-OH-dehydratase, and q-dihydropterin reductase, specifically in the absence of dehydratase activity. We conclude that formation of 7-substituted pterins in humans is a consequence of an absence of dehydratase activity, which might result from a genetic defect. A chemical mechanism for this rearrangement is presented. Our results also suggest that tetrahydroneopterin can be a cofactor for the phenylalanine hydroxylase system in vivo.

Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis

The chemical synthesis of [6- 3H]protopine is described. Microsomal preparations from Eschscholtzia californica cell suspension cultures challenged with a crude elicitor preparation from yeast, catalyse the hydroxylation of [6- 3H]protopine with the concomitant formation of [11- 3H]dihydrosanguinarine and HO 3H. This reaction served as an assay for the enzyme. The hydroxylase reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. Cytochrome c, inhibitors such as prochloraz and ketoconazole and carbon monoxide inhibit the hydroxylation reaction, suggesting that the enzyme is a cytochrome P-450 linked monooxygenase. The hydroxylase was induced by the yeast elicitor ca eight-fold, 20 hr after challenging the plant cell culture with the yeast carbohydrate, having no effect on the cell dry weight of the culture. The hydroxylase is specifically present in different plant species that produce benzo[c]phenanthridine alkaloids in culture and also respond to induction by the elicitor.

Ligninase-catalyzed hydroxylation of phenols

Abstract Lignin peroxidase (ligninase) catalyzes the hydroxylation of phenols in the presence of dihydroxyfumarate and molecular oxygen at pH 4 with a turnover number of 1.7 s −1 . After 3 h, 50% of a 2mM phenol solution reacts to give catechol and hydroquinone in a respective ratio of 4.5:1. The hydroxylase and dihydroxyfumarate oxidase reactions are strongly uncoupled; 10-fold more dihydroxyfumarate is oxidized, presumably to diketosuccinate, than phenol is hydroxylated. This hydroxylation reaction is distinct from all other reported reactions involving ligninase, in that hydrogen peroxide is not involved as the external oxidant. Hence the chemistries associated with ligninase compounds I and II are not involved in the hydroxylation reaction, although this cannot be ruled out for dihydroxyfumarate oxidation. Instead, superoxide and hydroxyl radicals are involved in the hydroxylation and ligninase compound III is an active intermediate. Phenol hydroxylation, therefore, is the first reported catalytically active role for ligninase compound III. In addition to phenol, p - and m -cresols, and l -tyrosine are also hydroxylation substrates.

The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens. The beta subunit is known to be identical with the enzyme protein disulphide-isomerase and to possess disulphide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer. We here report that lysyl hydroxylase, a homodimer, and algal prolyl 4-hydroxylase, a monomer, do not contain detectable protein disulphide-isomerase activity. Since the hydroxylase reaction mechanisms are similar, the data suggest that the protein disulphide-isomerase activity of the vertebrate prolyl 4-hydroxylase beta subunit is unlikely to be involved in the catalytic mechanism of the hydroxylation reaction.

Hyperphenylalaninemia in the hph-1 mouse mutant.

A mutation, resulting in a deficiency of liver GTP-cyclohydrolase activity, has been induced in the laboratory mouse. Mice homozygous for this mutation exhibit hyperphenylalaninemia under the following conditions: 1) early in life and 2) throughout life when exposed to phenylalanine. A phenylalanine loading regimen was used to discriminate between mutant and wild type mice on the basis of the resultant phenylalanine and tyrosine serum levels. Subjecting mice to this regimen reveals several distinguishing characteristics. Mutant mice exhibit approximately 2-fold higher peak phenylalanine levels than wild-type mice. In wild-type mice the hyperphenylalaninemic state is transient and rapidly abates while in mutant mice it is persistent and remains for a prolonged period. Mutant mice exhibit normal serum tyrosine levels after a loading challenge, while wild-type mice experience an increase in tyrosine levels. The loading regimen was also used to gauge the response of mutant hyperphenylalaninemic mice to exposure to chemical compounds required for normal phenylalanine catabolism (i.e. pteridine cofactors of the phenylalanine hydroxylase reaction). Mutant mice exposed to native enzyme cofactor or cofactor precursors exhibit a sharp decline in serum phenylalanine levels relative to their uninjected counterparts coupled with a tyrosine increase. By contrast, mutant mice exposed to nonprecursor compounds that are structurally related to the native cofactor, experience no diminution of serum phenylalanine levels.

A spectrophotometric assay for deacetoxycephalosporin C synthase

A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. K m values of 0.18 mM for penicillin N and 0.16 mM for α-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.

Modulation by pterins of the phosphorylation and phenylalanine activation of phenylalanine 4-mono-oxygenase.

The interaction between phenylalanine 4-mono-oxygenase and analogues of the natural cofactor (6R)-tetrahydrobiopterin [(6R)-BH4] was studied. The rate of cyclic AMP-dependent phosphorylation of phenylalanine 4-mono-oxygenase was inhibited only by those pterins [(6R)-BH4, (6S)-BH4 and 7,8-dihydrobiopterin (BH2)] that were able to decrease the potency and efficiency of phenylalanine as an allosteric activator of the hydroxylase. Since BH2 lacks cofactor activity, this was not required to modulate either the phosphorylation or the phenylalanine-activation of the hydroxylase. Half-maximal inhibition of the phosphorylation was observed at 1.9 microM-(6R)-BH4, 9 microM-(6S)-BH4 and 17 microM-BH2. Competition experiments indicated that all three pterins acted through binding to the cofactor site of the hydroxylase. Since the phosphorylation site and the cofactor binding site are known to reside, respectively, in the N- and C-terminal domains of the hydroxylase, the pterins were able to induce an interdomain conformational change. BH2, whose dihydroxypropyl group is not subject to epimerization, and (6S)-BH4 both inhibited the phosphorylation less efficiently than did the (6R)-epimer of BH4. Pterins with different spatial arrangements of the dihydroxypropyl side chain thus appeared to elicit different conformations of the phosphorylation site. The hydroxylase reaction showed a higher apparent Km for (6S)-BH4 than for (6R)-BH4 both when the native and the phenylalanine-activated enzyme were tested. For the activated enzyme Vmax was 40% lower with the (6S)-epimer than the (6R)-epimer, also when the more rapid enzyme inactivation occurring with the former cofactor was taken into account.

Tyrosinase isozyme heterogeneity in differentiating B16/C3 melanoma.

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.

Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida.

(2S)-Flavanone 3-hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)-naringenin to (2R,3R)-dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2-oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200-fold by a six-step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74,000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2-oxoglutarate, Fe2+ and ascorbate. With 2-oxo[1-14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)-(2R,3R)-dihydrokaempferol. (2S)-Naringenin, but not the (2R)-enantiomer is a substrate of the hydroxylase. (2S)-Eriodictyol is converted to (2R,3R)-dihydroquercetin. In contrast, 5,7,3',4',5'-pentahydroxy-flavanone is not a substrate. Apparent Michaelis constants for (2S)-naringenin and 2-oxoglutarate were determined to be respectively 5.6 mumol X l-1 and 20 mumol X l-1 at pH 8.5. The Km for (2S)-eriodictyol is 12 mumol X l-1 at pH 8.0. Pyridine 2,4-dicarboxylate and 2,5-dicarboxylate are strong competitive inhibitors with respect to 2-oxoglutarate with Ki values of 1.2 mumol X l-1 and 40 mumol X l-1, respectively.

Inhibition of rat liver microsomal cytochrome P-450 steroid hydroxylase reactions by imidazole antimycotic agents

The imidazole antimycotic agents ketoconazole, miconazole and clotrimazole were tested for their abilities to inhibit the reactions involved in the oxidative metabolism of androst-4-ene-3,17-dione by rat liver microsomal cytochromes P-450. All three compounds were found to function as potent inhibitors of steroid hydroxylase reactions, producing 50% inhibition of 6β-, 16β-, and 16α-hydroxylase activities at concentrations between 10 −7 and K −5M. The antimycotic agents, when added to liver microsomes, bound to cytochrome P-450 with high affinity to produce a “type II” spectral complex. These agents showed differential inhibition of the various steroid hydroxylases and were found not to affect the activities of the liver microsomal steroid 5α-reductase or the androst-4-ene-3,17-dione 17-oxidoreductase. The results presented demonstrate an interaction of these imidazole antimycotic agents with the various cytochromes P-450 of liver microsomes, resulting in selective inhibition of monooxygenase activity.

A paradoxical effect of hydralazine on prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts

The effect of hydralazine on several parameters of collagen biosynthesis has been studied in cultured human skin fibroblasts. Cells treated with hydralazine synthesized procollagen which was severely deficient in hydroxyproline and hydroxylysine, indicating inhibition of prolyl and lysyl hydroxylase reactions in the cell. Assays of prolyl and lysyl hydroxylase activities, however, revealed markedly increased levels in hydralazine-treated cells. The stimulatory effect of hydralazine could not be simulated in cell extracts, demonstrating its requirement for intact cells. The effect occurred slowly over a period of 96 h and was dependent on hydralazine concentration between 10 and 100 μ m. This phenomenon was also observed in lysyl hydroxylase-deficient mutants. In both normal and mutant cells the relative magnitude of the hydralazine effect could be modified by ascorbic acid in the culture medium. Ascorbic acid increased the response of prolyl hydroxylase to hydralazine from 1.5- to 2-fold to 3- to 7-fold, whereas it decreased the response of lysyl hydroxylase to hydralazine from 4- to 8-fold to 2- to 3-fold. Total collagen synthesis was substantially reduced in hydralazine-treated cells; the time course and the dose-response relationship were similar to those observed for the hydroxylases. α,α′-Dipyridyl, an iron chelator, mimicked these effects of hydralazine. The studies suggest the existence in cultured cells of a compensatory mechanism for overproduction of these crucial enzymes in collagen biosynthesis, a mechanism which remains functional in cells derived from patients afflicted with hydroxylysine-deficient collagen disease.

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H 2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H 2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H 2O with the hydroxyl proton of isoamyl alcohol. It is the [ 3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H 2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.

Markedly different ascorbate dependencies of the sequential alpha-ketoglutarate dioxygenase reactions catalyzed by an essentially homogeneous thymine 7-hydroxylase from Rhodotorula glutinis.

The alpha-ketoglutarate dioxygenase, thymine 7-hydroxylase (EC 1.14.11.6), has been purified from cultures of Rhodotorula glutinis grown with thymine as a nitrogen source. The purification scheme developed yielded essentially homogeneous preparations of the 7-hydroxylase and also purified another alpha-ketoglutarate dioxygenase, pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3). The purity of the 7-hydroxylase was determined with analytical disc gel electrophoresis in which runs were varied with respect to pH, extent of cross-linking, and the presence of sodium dodecyl sulfate-mercaptoethanol. The 7-hydroxylase apparently exists as a monomer since its molecular weight was 42,700 when determined by molecular gel filtration chromatography and was 40,300 when determined by analytical disc gel electrophoresis under denaturing conditions. Gel filtration chromatography under nondenaturing conditions was used to show that the 2'-hydroxylase has a molecular weight of 64,600. The essentially homogeneous preparations of the 7-hydroxylase were shown to catalyze the thymine-, 5-hydroxymethyluracil-, and 5-formyluracil-dependent oxygenations that are coupled to the decarboxylation of alpha-ketoglutarate, as well as a putative uncoupled decarboxylation which is dependent on uracil. Furthermore, these enzyme preparations were used to show that ATP stimulated the 7-hydroxylase reaction in the absence of ascorbate. Even though it is attractive to consider the four pyrimidine-dependent reactions as being catalyzed by the same active site, they were shown to differ markedly in their dependencies on ascorbate or ATP. The effects of ascorbate and ATP on these reactions, and on the 2'-hydroxylase reaction, are discussed in terms of the possible roles of ascorbate and ATP.

Induction and characterization of a microsomal flavonoid 3'-hydroxylase from parsley cell cultures.

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.

The source of oxygen in the reaction catalysed by collagen lysyl hydroxylase

The synthetic peptides (Pro-Pro-Gly)5 and (Ile-Lys-Gly)5-Phe were hydroxylated with collagen prolyl hydroxylase and lysyl hydroxylase in an 18O2 atmosphere. The oxygen atoms in the hydroxy groups of hydroxyproline and hydroxylysine were 87% and 6.5% respectively derived from the atmospheric 18O2. The results are consistent with those reported previously for proline hydroxylation in vivo [Fujimoto & Tamiya (1962) Biochem. J. 84, 333-335; Prockop, Kaplan & Udenfriend (1962) Biochem. Biophys. Res. Commun. 9, 192-196; Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501; Prockop, Kaplan & Udenfriend (1963) Arch. Biochem. Biophys. 101, 499-503] and in vitro [Cardinale, Rhoads & Udenfriend (1971) Biochem. Biophys. Res. Commun. 43, 537-543] and for lysine hydroxylation in vivo [Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501]. In view of the similarities of these two oxygenase-type hydroxylation reactions the participation of intermediates is proposed, the oxygen atoms of which are exchangeable with those of water. The atmospheric oxygen atoms incorporated into the intermediate must be equilibrated with water oxygen atoms in the slower lysyl hydroxylase reaction.

Metabolism of radiotestosterone to 3β,6α-and 3β,7α-dihydroxy 5α-steroids by rat ventral, canine and human prostate in organ culture

We report here the structural assignment for the hydroxylated 5α-reduced metabolites in the culture medium following incubation of radiolabelled testosterone with explants of rat ventral prostate, canine cauda epididymidis and prostate, and benign hyperplastic canine and human prostate. Explants were incubated for 21 h at 37°C in surface contact with serum-free Trowell's T8 medium containing 1.7 μM or 8.5 nM substrate. After uptake determination, radiosteroid patterns in explants and media were obtained by t.l.c. The C 19O 3-metabolites released into the culture medium were resolved by h.p.l.c. and fractions migrating with appropriate reference compounds were crystallized to constant SA with carriers synthesized for this purpose. 5α-Androstane-3β,6α,17β-triol and 3β, 6α-dihydroxy-5α-androstan-17-one were identified as the major C 19C 3-radiometabolites of rat ventral prostate. In the culture medium of canine prostate and epididymis and human prostate, 5α-androstane-3β,7α,17β-triol and 3β,7α-dihydroxy 5α-androstan-17-one were the principal hydroxylation products, with 6α-hydroxy epimers as significant minor products of the canine prostate and epididymis and 5α-androstane-3β,7β,17β-triol as a significant minor radiometabolite of human prostate tissue. Treatment of the castrated dog with androgen and estrogen causes an oxidative shift to striking predominance of 3β,7α-dihydroxy 5α-androstan-17-one. The 3β-hydroxy-5α-androstane configuration of the identified C 19O 3-metabolites supports a pathway of prostatic and epididymal testosterone disposition which effects activation by 5α-reductase and inactivation and egress by the coupled 5α-3-oxosteroid reductase/3β-hydroxysteroid hydroxylase reactions.

Two forms of adrenodoxin reductase from mitochondria of bovine adrenal cortex.

Two forms of adrenodoxin reductase, differing in isoelectric properties and thermal stability, have been highly purified from bovine adrenal cortex mitochondria. Both preparations appear to be homogeneous based upon polyacrylamide disc‐gel electrophoresis, isoelectric focusing, and ion‐exchange chromatography. Both are monomeric proteins containing one FAD per molecule (Mr 50000) and exhibit spectra typical of flavoproteins. The two preparations are indistinguishable by their amino acid compositions and N‐terminal (serine) and C‐ter‐ minal (leucine) amino acids, but reveal distinct sugar contents. Electrophoretic mapping of the protease digests of the two preparations indicates that the two have identical, or closely similar, primary structures.Each form functions equally as an adrenodoxin‐dependent electron transport component from NADPH to the P‐450 cytochromes in the two different reconstituted steroid monooxygenase systems which catalyze cholesterol side‐chain cleavage reaction and steroid 11β‐(18‐)hydroxylase reaction, respectively. In all adrenodoxin reductase‐ containing fractions from adrenal cortex so far examined, i.e. the soluble fraction, and the sonic‐solubilized and detergent‐solubilized mitochondrial fractions, a constant ratio of about 2:5 between the two forms is found as judged by the DEAE‐cellulose chromatogram. No evidence has been obtained for mutual conversion of the two forms of the reductase, or for dissociation of the sugar moiety during the purification process and under various conditions examined.

Uncoupling in the γ-butyrobetaine hydroxylase reaction by [formula omitted]- and [formula omitted]-carnitine

In the presence of D = -carnitine significant decarboxylation of 2-oxoglutarate occurs with γ-butyrobetaine hydroxylase (EC 1.14.11.1) both from Pseudomonas sp AK 1 and from human kidney. No product was formed from carnitine when D = L = -carnitine was incubated with either enzyme but succinate was formed in 1:1 stoichiometry to decarboxylation using D = -carnitine and the human enzyme. L = -Carnitine is also an uncoupler for the human enzyme. There is no significant decarboxylation of 2-oxoglutarate in the absence of a substrate, but during normal catalysis in the presence of γ-butyrobetaine the formation of CO 2 from 2-oxoglutarate exceeds carnitine formation with 20% for the human enzyme.

Effect of homologous antistreptococcal antibodies on testicular tissue

Nonachlazin (6 mg/kg) and indomethacin (10 mg/kg) intraperitoneally have a unidirectional effect on the activity of tyrosin hydroxylase, reducing it in the hypothalamus and in the brain stem and increasing it in the heart septum. In the brain, the effects of nonachlazin and indomethacin are not summarized whereas in the heart their effects are summarized. Unlike indomethacin, nonachlazin exerts a direct effect on the rate of tyrosin hydroxylase reaction but has no effect on the activity of cyclooxygenase and on synthetase of prostaglandins. It is concluded that prostaglandins produce a modulating action on the activity of brain and heart tyrosine hydroxylase and participate in the mechanism of action of nonachlazin.

Oxygenation of a carbinol carbon in the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction

1. 1. The pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3) reaction of Neurospora crassa was studied with uracil-β- d-arabinofuranoside (AraU) as the nucleoside substrate. 2. 2. That the 2'-carbinol carbon of AraU was oxygenated is indicated by the stoichiometry of the utilization of AraU and formation of products, the requirement for molecular oxygen, the stimulations effected by Fe 2+, ascorbate and catalase and the substrate specificity of the 2'-hydroxylase reaction. 3. 3. The parallel loss of the AraU and deoxyuridine-dependent activities upon heat denaturation, their copurification and the lower levels of both activities in a mutant strain deficient in the 2'-hydroxylase indicate that the same enzyme catalyzed the AraU and deoxyuridine-dependent reactions.