Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori.

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.     

Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.     

Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives.

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 mm) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 mm) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 mm OMP and 2 mm uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mm OMP and 1 mm 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.     

Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicylatoxidans

In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and (1)H and (13)C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3- and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.     

Geranyl pyrophosphate synthase: characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis)

Cell-free homogenates from sage (Salvia officinalis) leaves convert dimethylallyl pyrophosphate and isopentenyl pyrophosphate to a mixture of geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate, with farnesyl pyrophosphate predominating. These prenyltransferase activities were localized primarily in the soluble enzyme fraction, and separation of this preparation on Sephadex G-150 revealed the presence of a partially resolved, labile geranyl pyrophosphate synthase activity. The product of the condensation reaction between [1-14C]dimethylallyl pyrophosphate and [1-3H]isopentenyl pyrophosphate was verified as [14C,1-3H]geranyl pyrophosphate by TLC isolation, enzymatic hydrolysis to geraniol, degradative studies, and the preparation of the crystalline diphenylurethane. The cis-isomer, neryl pyrophosphate, was not a product of the enzymatic reaction. By employing a selective tissue extraction procedure, the geranyl pyrophosphate synthase activity was localized in the leaf epidermal glands, the site of monoterpene biosynthesis, suggesting that the role of this enzyme is to supply the C10 precursor for the production of monoterpenes. Glandular extracts enriched in geranyl pyrophosphate synthase were partially purified by a combination of hydrophobic interaction chromatography on phenyl-Sepharose and gel permeation chromatography on Sephadex G-150. Substrate and product specificity studies confirmed the selective synthesis of geranyl pyrophosphate by this enzyme, which was also characterized with respect to molecular weight, pH optimum, cation requirement, inhibitors, and kinetic parameters, and shown to resemble other prenyltransferases.     

Lipoxygenase from potato tubers. Partial purification and properties of an enzyme that specifically oxygenates the 9-position of linoleic acid

A lipoxygenase (EC 1.13.1.13) was partially purified from potato tubers and was shown to differ from previously characterized soya-bean lipoxygenases in the positional specificity and pH characteristics of the oxygenation reaction. The potato enzyme converted linoleic acid almost exclusively (95%) into 9-d-hydroperoxyoctadeca-trans-10,cis-12-dienoic acid. The 13-hydroperoxy isomer was only a minor product (5%). Linolenic acid was an equally effective substrate, which was also oxygenated specifically at the 9-position. The enzyme had a pH optimum at 5.5-6.0 and was inactive at pH9.0. A half-maximal velocity was obtained at a linoleic acid concentration of 0.1mm. No inhibition was observed with EDTA (1mm) and cyanide (1mm) or with p-chloromercuribenzoate (0.2mm). Haemoproteins were not involved in the lipoxygenase activity. The molecular weight of the enzyme was estimated from gel filtration to be approx. 10(5). Preliminary evidence suggested that the enzyme oxygenated the n-10 position of fatty acids containing a penta(n-3, n-6)diene structure.     

The bacterial degradation of flavonoids. Hydroxylation of the A-ring of taxifolin by a soil pseudomonad

Cell-free extracts prepared from a Pseudomonas sp. grown on (+)-catechin oxidized taxifolin in the presence of NAD(P)H and molecular oxygen. The enzyme catalysing the reaction was partially purified and shown to be a flavoprotein. The product was identified as 3',4',5,7,8-pentahydroxyflavanonol (dihydrogossypetin).     

Purification and properties of sulfoacetaldehyde sulfo-lyase, a thiamine pyrophosphate-dependent enzyme forming sulfite and acetate.

Sulfoacetaldehyde sulfo-lyase, which decomposes sulfoacetaldehyde to sulfite and acetate, was extracted from a bacterium grown on taurine, and purified, and characterized. A method for assay of enzyme activity was devised on formation of a bisulfite adduct with benzaldehyde. The enzyme was purified 14-fold from an extract of cells grown on taurine and appeared homogeneous on disc-electrophoresis. The molecular weight of the enzyme was estimated to be 85,000 by gel filtration. The enzyme required thiamine pyrophosphate (TPP) and Mg2+ for activity and preincubation with TPP and Mg2+ was required for maximum activity. The optimum pH for activity was 7.5. The Km value for TPP was determined to be 2.7 muM and that for sulfoacetaldehyde to be 5.0mM. Sulfite was produced only from sulfoacetaldehyde among a variety of sulfonates tested. rho-Chloromercuribenzoate, EDTA, and sulfite, a reaction product, inhibited the enzyme reaction. The enzyme seemed to be inducible, since activity was found in extracts of cells grown on taurine but not on peptone.     

3-Dehydroquinate synthase in germinating Phaseolus mungo seedlings.

Dehydroquinate synthase, an enzyme catalyzing the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, was detected in cell-free extracts of etiolated Phaseolus mungo seedlings. The reaction product, 3-dehydroquinate, formed from [1-14C]DAHP was identified by paper-radiochromatography. The enzyme required NAD+ and Co2+ for activity.     

The biosynthesis of ovothiol A (N-methyl-4-mercaptohistidine). Identification of S-(4'-L-histidyl)-L-cysteine sulfoxide as an intermediate and the products of the sulfoxide lyase reaction.

Crude extracts of Crithidia fasciculata catalyse the formation of 4-mercapto-L-histidine, an intermediate in the biosynthesis of ovothiol A (N1-methyl-4-mercaptohistidine), in the presence of histidine, cysteine, Fe2+ and pyridoxal phosphate. This activity was present in a 35-55% ammonium sulfate fraction that was shown to produce a transsulfuration intermediate in the absence of pyridoxal phosphate. The transsulfuration intermediate was isolated and identified as S-(4'-L-histidyl)-L-cysteine sulfoxide. The synthase activity, partially purified by anion-exchange chromatography, was shown to require oxygen and could be used to synthesize a number of isotopically labeled S-(4'-L-histidyl)-L-cysteine sulfoxides. Sulfoxide lyase activity was partially resolved from the synthase by anion-exchange chromatography. The phenylhydrazone of the product derived from the cysteine moiety of the sulfoxide coeluted with the phenylhydrazone of pyruvate on HPLC, but this assignment could not be confirmed by mass spectral analysis. S-(4'-[14C]L-histidyl)-[U-13C3,15N]L-cysteine sulfoxide was synthesized and converted to products of the lyase reaction in the presence of lactate dehydrogenase and NADH. The 13C-labeled product was identified by 13C-NMR spectroscopy as lactate and the primary product of the lyase reaction is therefore pyruvate. With S-(4'[3H]L-histidyl)-[14C]L-cysteine sulfoxide as the substrate [14C]lactate, [14C]cysteine and [3H]4-mercaptohistidine could be detected as products of the lyase reaction, but the sum of the two thiol species exceeded the amount of sulfoxide substrate used. Evidence is presented that this anomaly was due to the utilization of sulfur from dithiothreitol for the formation of cysteine.     

Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

Purification and properties of GTP-cyclohydrolase of the yeast Pichia guilliermondii

GTP-cyclohydrolase was isolated from the Fe-deficient cells of Pichia guilliermondii and purified 440-fold by treatment of extracts with streptomycin sulfate as well as by protein fractionation with (NH4)2SO4 at 25-45% saturation, gel filtration through Sephadex G-200 and DEAE-cellulose chromatography. The curves for the dependence of specific activity of GTP-cyclohydrolase on substrate and cofactor concentrations are non-hyperbolic; the values of [S]0.5 for GTP and Mg2+ are 2.2 X 10(-5) and 2 X 10(-4) M, respectively. The enzyme activity is inhibited by pyrophosphate ([I]0.5 = 5.8 X 10(-4) M), orthophosphate ([I]0.5 = 4.5 X 10(-3) M), heavy metal ions and chelating agents. The temperature optimum for the enzyme activity lies at 42-45 degrees C. The enzyme is labile at 4 degrees C but can well be stored at -15 degrees C. The pyrimidine product of the cyclohydrolase reaction, 2.5-diamino-6-oxy-4-ribosyl-aminopyrimidine-5'-phosphate, as well as pyrophosphate were purified from the reaction medium and identified.     

Canine thyroid fucokinase.

A radiometric assay was developed for fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) based on the conversion of L-[14C]fucose to L-[14C]fucose 1-phosphate which is trapped and counted on ion exchange paper. This assay was used to detect the presence of a fucokinase in canine thyroid tissue which was subsequently purified 2754-fold over the crude tissue extracts. The product of the fucokinase was identified as the beta-anomer. The pH versus activity curve for the enzyme appears biphasic with optima at pH 6.5 and pH 8.25. The enzyme was shown to be highly specific for L-fucose with a Km of 2.6 - 10(-5) M at pH 8.25. It was shown to be absolutely specific for ATP as a phosphate donor with a Km of 6.3 - 10(-4) M at pH 8.25. The enzyme requires a divalent cation. Mg2+ is slightly more effective than Mn2+ in meeting this need. The molecular weight of the enzyme has been determined to be 494 000 +/- 12 400.     

Crystalline hemoprotein from Pseudomonas that catalyzes oxidation of side chain of tryptophan and other indole derivatives.

A new enzyme which catalyzes the oxidation of the side chain of tryptophan and other indole derivatives, has been purified to apparent homogeneity from Pseudomonas and crystallized. The overall purification was about 25-fold with a yield of 4.5%. The purified enzyme was apparently homogeneous as judged by polyacrylamide gel electrophoresis. The molecular weight estimated by gel filtration was approximately 280,000 and sedimentation coefficient (S20,w) was 11 by sucrose density gradient ultracentrifugation. The absorption spectra indicated that the enzyme was a hemoprotein. The purified enzyme was shown to catalyze the reaction in which 1 mol each of NH3 and CO2 was formed at the expense of 1 mol each of L-tryptophan and molecular oxygen. Neither peroxidase nor catalase activity was detected in the purified enzyme and no formation of H2O2 was observed during the enzyme reaction. The product(s) of the reaction was unstable but was converted to and was identified as its stable quinoxaline derivative, 2-(3-indolyl)quinoxaline, in the presence of o-phenylenediamine. These results indicate that the product of the reaction was 3-indolylglycoaldehyde or 3-indolylglyoxal. A variety of other indole derivatives such as D-tryptophan, 5-hydroxyl-L-tryptophan, tryptamine, serotonin, melatonin, N-acetyl-L-tryptophan, N-acetyl-L-tryptophanamide, 3-indoleacetamide, 3-indolelactic acid, 3-indolepropionic acid, 3-indoleethanol, and skatole were also substrates.     

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.     

The formation of 5-phosphomevalonate by mevalonate kinase in Hevea brasiliensis latex

1. Evidence has been produced for the formation of 5-phosphomevalonate from potassium dl-mevalonate by the latex of Hevea brasiliensis and by reconstituted freeze-dried serum obtained from this latex. 2. The enzyme, mevalonate kinase, catalysing the formation of 5-phosphomevalonate from potassium dl-mevalonate and ATP has been partially purified. 3. 5-Phosphomevalonate formed by the purified mevalonate kinase from potassium [2-(14)C]mevalonate has been shown to be incorporated by latex into rubber to about 2.4 times the extent of dl-mevalonate. 4. The enzyme can utilize inosine triphosphate as effectively as adenosine triphosphate as a phosphate donor and is also slightly active with uridine triphosphate. 5. The enzyme was fairly stable to a range of pH values and temperatures, the activity being optimum at pH7.5 and 60-70 degrees . The energy of activation was 10.7kcal./mole. The K(m) values were 0.13mm for potassium dl-mevalonate and 2.0mm for ATP at 30 degrees . 6. The enzyme required the presence of Mn(2+) (1mm) for maximum activity; this could be replaced by Mg(2+) (4mm), which was less effective, and by Ca(2+), which was far less effective. 6. Although the enzyme did not require cysteine or reduced glutathione for activation in aerobic conditions, it was inhibited by reagents known to react with thiol groups.     

Glycerol metabolism in higher plants: glycerol kinase

Glycerol kinase activity was identified in extracts of higher plant seeds and seedlings, and was partially purified and characterized from cucumber radicle tissue. The enzyme was localized in the post-mitochondrial supernatant of the cell, and catalyzed the formation of glycerol-3-phosphate. The pH optiumum was 9.0. ATP, CTP, GTP or UTP could be used as the phosphoryl group donor. The Km for glycerol was 55 microM and Km values for the nucleoside triphosphates were 145-620 microM. The Vmax for the reaction was 40-78 pmol product per min. Kinetic data indicate that the enzyme has a sequential mechanism.     

The distinctiveness of ATP:citrate lyase from Aspergillus nidulans

ATP:citrate lyase (ACL), an important enzyme in lipid synthesis, has been purified from Aspergillus nidulans to a specific activity of 19.6 micromol min(-1) mg(-1), almost twice that of any other purified ACL and shown to be distinct from any previously purified ACL. The enzyme is a 371+/-31 kDa hexamer of 3 alpha, 3 beta proteins, unlike the 4 alpha tetramer found in rats or yeasts. The molecular weights of the alpha and beta protein subunits were determined by SDS-PAGE to be 70 and 55 kDa.ACL in A. nidulans (unlike Aspergillus niger) appears to be regulated by the carbon source present in the media. In crude extracts, it was found at high activity (88 micromol min(-1) mg protein(-1)) in glucose-grown cells but only at low activity (10 micromol min(-1) mg protein(-1)) in acetate-grown cells.     

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.     

Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme

We report the identification and purification of a novel enzyme from soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reaction is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase. The enzyme was purified from crude extracts of soybean root nodules approximately 100-fold to apparent homogeneity with a final specific activity of 10 micromol/min/mg. The enzyme exhibited a native molecular mass of approximately 68 kDa by gel filtration chromatography and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 +/- 2 kDa. The purified enzyme obeyed normal Michaelis-Menten kinetics, and the K(m) for 5-hydroxyisourate was determined to be 15 microM. The amino-terminal end of the purified protein was sequenced, and the resulting sequence was not found in any available data bases, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin.