N5, N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming) from the extreme thermophile Methanopyrus kandleri

A bacterium tentatively classified as Arthrobacter strain Py1 being capable to degrade pyrrole-2-carboxylate as only source of carbon, nitrogen, and energy was isolated from soil. In contrast to many other N-heterocyclic compounds, growth of the isolate on pyrrole-2-carboxylate was not affected by molybdate or its specific inhibitor tungstate, indicating a molybdoenzyme-independent breakdown. The latter was initiated by a hydroxylation reaction catalyzed by a pyrrole-2-carboxylate oxygenase, which also exhibited an NADH-cytochrome c reductase activity. The pyrrole-2-carboxylate oxygenase reaction as examined in cell extracts depended on NADH, FAD, and pyrrole-2-carboxylate; the apparent K _m values were 44, 6, and 43 M, respectively. A degradation pathway for pyrrole-2-carboxylate is proposed which involves 5-hydroxy-pyrrole-2-carboxylate and 2-oxoglutarate.     

Formation and excretion of pyrrole-2-carboxylic acid. Whole animal and enzyme studies in the rat.

A corrected method for the measurement of pyrrole-2-carboxylate in rat urine was used in studies of its excretion under various experimental conditions. The findings implicated administered hydroxy-L-proline as a relatively efficient source of urinary pyrrole-2-carboxylate and tended to exclude administered L-proline as a significant direct source. Removal of aerobic gut flora had no influence on the excretion of pyrrole-2-carboxylate either endogenously or following hydroxy-L-proline administration. Related studied showed that rat kidney L-amino acid oxidase catalyzes oxidation of hydroxy-L-proline to delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate on acidification of reaction mixtures. All findings were consistent with hydroxy-L-proline as the source of endogenous pyrrole-2-carboxylate excretion. Excretion patterns and labeling patterns were compared after administration of pyrrole-2-carboxylate or of hydroxy-proline epimers. From these data, the true excretion product of hydroxy-L-proline oxidation by L-amino acid oxidase appeared to be the unstable oxidation product, delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate in urine. The capacity of homogenates of guinea pig kidney and human kidney to carry out oxidation of hydroxy-L-proline to pyrrole-2-carboxylate was much less than that of rat kidney, consistent with the lower levels of urinary pyrrole-2-carboxylate in these species. Experiments designed to examine the modest increase of pyrrole-2-carboxylate excretion after proline loads led to new observations on tissue levels of hydroxy-L-proline following proline administration and on the inhibition by L-proline of hydroxy-L-proline oxidase.     

Biosynthesis of the nargenicin A<Subscript>1</Subscript> pyrrole moiety from <Emphasis Type="Italic">Nocardia</Emphasis> sp. CS682

A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from l-proline. Nargenicin A₁, a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A₁ in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A₁ was also derived from l-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A₁ is initiated by NgnN4 that catalyzes ATP-dependent activation of l-proline into l-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert l-proline into pyrrole-2-carboxylic acid moiety.     

Novel reversible indole-3-carboxylate decarboxylase catalyzing nonoxidative decarboxylation

After enrichment culture with indole-3-carboxylate in static culture, a novel reversible decarboxylase, indole-3-carboxylate decarboxylase, was found in Arthrobacter nicotianae FI1612 and several molds. The enzyme reaction was examined in resting-cell reactions with A. nicotianae FI1612. The enzyme activity was induced specifically by indole-3-carboxylate, but not by indole. The indole-3-carboxylate decarboxylase of A. nicotianae FI1612 catalyzed the nonoxidative decarboxylation of indole-3-carboxylate into indole, and efficiently carboxylated indole and 2-methylindole by the reverse reaction. In the presence of 1 mM dithiothreitol, 50 mM Na2 S2O3, and 20% (v/v) glycerol, indole-3-carboxylate decarboxylase was partially purified from A. nicotianae FI1612. The purified enzyme had a molecular mass of approximately 258 kDa. The enzyme did not need any cofactor for the decarboxylating and carboxylating reactions.     

Exploratory studies on azole carboxamides as nucleobase analogs: thermal denaturation studies on oligodeoxyribonucleotide duplexes containing pyrrole-3-carboxamide.

In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide, was synthesized by a new route from the ester, methyl 1-(2'-deoxy-3',5'-di-O-p -toluoyl-beta-D-erythro-pentofuranosyl)pyrrole-3-carboxylate, obtained from the coupling reaction between 1-chloro-2-deoxy-3,5-di-O -toluoyl-d-erythropentofuranose and methyl pyrrole-3-carboxylate by treatment with dimethylaluminum amide. 1-(2'-Deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide was incorporated into a series of oligodeoxyribonucleotides by solid-phase phosphoramidite technology. The corresponding oligodeoxyribonucleotides with 3-nitropyrrole in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that pyrrole-3-carboxamide, which could conceptually adopt either a dA-like or a dI-like hydrogen bond conformation, pairs with significantly higher affinity to T than to dC. Pyrrole-3-carboxamide further resembles dA in the relative order of its base pairing preferences (T >dG >dA >dC). Theoretical calculations on the model compound N-methylpyrrole-3-carboxamide using density functional theory show little difference in the preference for a syntau versus anti conformation about the bond from pyrrole C3 to the amide carbonyl. The amide groups in both the minimized antitau and syntau conformations are twisted out of the plane of the pyrrole ring by 6-14 degrees. This twist may be one source of destabilization when the amide group is placed in the helix. Another contribution to the difference in stability between the base pairs of pyrrole-3-carboxamide with T and pyrrole-3-carboxamide with C may be the presence of a hydrogen bond in the former involving an acidic proton (N3-H of T).     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.     

Polyol-pathway enzymes of human brain. Partial purification and properties of sorbitol dehydrogenase.

Sorbitol dehydrogenase was isolated from human brain and purified 690-fold, giving a final specific activity of 11.1 units/mg of protein. The enzyme preparation was nearly homogeneous, but was unstable at most temperatures. It exhibited a broad pH optimum of 7.5-9.0 in the forward reaction (i.e. sorbitol leads to fructose), and of 7.0 in the reverse reaction (i.e. fructose leads to sorbitol). Substrate-specificity studies demonstrated that the enzyme had the capability to oxidize a wide range of polyols and that the enzyme had a higher affinity for substrates in the forward reaction than in the reverse reaction, e.g. Km for sorbitol was 0.45 mM, and that for fructose was 480 mM. However, the Vmax. was 10 times greater in the reverse reaction. At high concentrations of fructose (500 mM) the enzyme exhibited substrate inhibition in the reverse reaction. The enzyme mechanism was sequential, as determined by the kinetic patterns arising from varying the substrate concentrations. In addition, both fructose and NADH protected the enzyme against thermal inactivation. These findings, together with product-inhibition data, suggested that the mechanism is random rapid equilibrium with two dead-end complexes.     

Enzymatic formation of uridine diphosphate N-acetyl-D-mannosamine.

An enzyme that catalyzes the interconversion of UDP-N-acetyl-D-glucosamine and UDP-N-acetyl-D-mannosamine was purified about 700-fold from the supernatant fraction of Bacillus cereus, and the properties of this enzyme were studied. This enzyme was not stimulated by NAD+, NADH, or any metal ions. The optimum pH was between 7.5 and 8.0. At equilibrium of the reaction, the ratio of UDP-N-acetylglucosamine to UDP-N-acetylmannosmaine was about 9:1. The enzyme was inactive toward free N-acetylhexosamines, their phosphate esters, UDP-glucose, and UDP-N-acetylgalactosamine. A stimulatory role of UDP-N-acetylglucosamine was demonstrated. In the reaction with UDP-N-acetylglucosamine, the rate as a function of substrate concentration showed a sigmoidal relationship with a Hill coefficient of 1.8 and an apparent Km value for UDP-N-acetylglucosamine of 1.1 mM. The reverse reaction with UDP-N-acetylmannosamine required the presence of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine concentration required for half-maximal activation was about 0.5 mM. The apparent Km for UDP-N-acetylmannosamine measured in the presence of 0.5 mM UDP-N-acetylglucosamine was 0.22mM. Other nucleotides or hexosamine derivatives were not stimulatory. The same activity was found in cell extracts from several bacterial species.     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Purification and catalytic properties.

5-Oxo-L-prolinase, an enzyme that catalyzes the conversion of 5-oxo-L-proline (L-pyroglutamate; L-2-pyrrolidone-5-carboxylate) to L-glutamate coupled with the cleavage of ATP to ADP and Pi, has been purified about 1600-fold from rat kidney. Purification was carried out in the presence of 5-oxo-L-proline which protects the enzyme under a variety of conditions. An estimate of the molecular weight (about 325,000) was made by gel filtration on Sephadex G-200. K+ (or NH4+) and Mg2+ were required for activity. GTP, ITP, CTP, and UTP were much less active than ATP; dATP was 43% as active as ATP. ADP inhibited and addition of pyruvate kinase and phosphoenolpyruvate activated the reaction. The enzyme, which is protected during storage by dithiothreitol, is inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetamide. The apparent Km values for 5-oxo-L-proline and ATP are, respectively, 0.05 and 0.17 mM. The pH profile indicates a broad range of activity from about pH 5.5 to pH 11.2 with apparent maxima at about pH 7 and pH 9.7. The formation of Pi and glutamate was equimolar over a wide pH range. When the enzyme was incubated with ATP, Mg2+, K+, and L-2-imidazolidone-4-carboxylate or L-dihydroorotate, cleavage of ATP to ADP and Pi occurred, but no cleavage of the imino acid substrates was observed; when the enzyme was incubated under these conditions with 2-piperidone-6-carboxylate, 4-oxy-5-oxoproline, and 3-oxy-5-oxoproline, the corresponding dicarboxylic amino acids were formed, but the molar ratio of Pi to amino acid formation was significantly greater than unity.     

Purification and properties of 4-hydroxycyclohexanecarboxylate dehydrogenase from Corynebacterium cyclohexanicum

4-Hydroxycyclohexanecarboxylate dehydrogenase, which requires NAD as a cofactor, was detected in crude soluble extracts of Corynebacterium cyclohexanicum grown on cyclohexanecarboxylic acid as the sole carbon source. The dehydrogenase was purified from extracts to an electrophoretically homogenous state by ammonium sulfate precipitation and chromatography on DEAE-650s, agarose-NAD and hydroxyapatite. The enzyme consisted of two identical subunits and had a native relative molecular mass of 53,600. There were two residues each of cysteine and tryptophan in the enzyme molecule. Oxo acid rather than hydroxy acid was routinely used as substrate for assay of the enzyme. The enzyme is highly specific for 4-oxocyclohexanecarboxylic acid: the carboxyl group is essential and the position of carbonyl group is important; neither the 2-oxo nor the 3-oxo homologue was used as substrate. A methyl substitution on the ring of 4-oxocyclohexanecarboxylate resulted in an almost complete loss of its activity. The reduction product was identified as trans-4-hydroxycyclohexanecarboxylic acid by gas-liquid chromatography and mass spectrometry. It was used as a substrate for the reverse reaction in the presence of NAD but not its cis-isomer. The enzyme was specific for the B-side (pro-S) hydrogen of NADH in the hydrogen transfer from NADH to 4-oxocyclohexanecarboxylate. The Km values for 4-oxocyclohexanecarboxylate and NADH in the reduction reaction at pH 6.8 were 0.50 mM and 0.28 mM, respectively, whereas those for trans-4-hydroxycyclohexanecarboxylate and NAD in the oxidation reaction at pH 8.8 were 0.51 mM and 0.23 mM, respectively. The equilibrium constant of the reaction was 1.79 x 10(-10) M. The enzyme was strongly inhibited by N-bromosuccinimide.     

Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850?min(-1) and a K (m) of 540?μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35?°C, 4% (v/v) ethanol, 2,500?U ml(-1) (8.3?mg?ml(-1)) of enzyme, and 40?g l(-1) oleic acid. Under these conditions, 40?g l(-1) (142?mM) oleic acid was converted into 40?g l(-1) (133?mM) 10-hydroxystearic acid for 150?min, with a molar yield of 94% and a productivity of 16?g l(-1)?h(-1), and olive oil hydrolyzate containing 40?g l(-1) oleic acid was converted into 40?g l(-1) 10-hydroxystearic acid for 300?min, with a productivity of 8?g l(-1)?h(-1).     

Characterization of vanadium bromoperoxidase from Macrocystis and Fucus: reactivity of vanadium bromoperoxidase toward acyl and alkyl peroxides and bromination of amines

Vanadium bromoperoxidase (V-BrPO) has been isolated and purified from the marine brown algae Fucus distichus and Macrocystis pyrifera. V-BrPO catalyzes the oxidation of bromide by hydrogen peroxide, resulting in the bromination of certain organic acceptors or the formation of dioxygen. V-BrPO from F. distichus and M. pyrifera have subunit molecular weights of 65,000 and 74,000, respectively, and specific activities of 1580 units/mg (pH 6.5) and 1730 units/mg (pH 6) for the bromination of monochlorodimedone, respectively. As isolated, the enzymes contain a substoichiometric vanadium/subunit ratio; the vanadium content and specific activity are increased by addition of vanadate. V-BrPO (F. distichus, M. pyrifera, and Ascophyllum nodosum) also catalyzes the oxidation of bromide using peracetic acid. In the absence of an organic acceptor, a mixture of oxidized bromine species (e.g., hypobromous acid, bromine, and tribromide) is formed. Bromamine derivatives are formed from the corresponding amines, while 5-bromocytosine is formed from cytosine. In all cases, the rate of the V-BrPO-catalyzed reaction is much faster than that of the uncatalyzed oxidation of bromide by peracetic acid, at pH 8.5, 1 mM bromide, and 2 mM peracetic acid. In contrast to hydrogen peroxide, V-BrPO does not catalyze formation of dioxygen from peracetic acid in either the presence or absence of bromide. V-BrPO also uses phenylperacetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid to catalyze the oxidation of bromide; dioxygen is not formed with these peracids. V-BrPO does not catalyze bromide oxidation or dioxygen formation with the alkyl peroxides ethyl hydroperoxide, tert-butyl hydroperoxide, and cuminyl hydroperoxide.     

Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(- 1) and k(cat)/K(M) of 2.5.10(5) M(- 1) s(- 1). The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (K(I)s of 21-78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K(I)s around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K(I)s in the range of 10-90 microM. It is difficult to explain the inhibitory activity of carbonate (K(I) of 10 microM) against hCA III, also considering the fact that this ion has an affinity of 15-73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (K(I) of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).     

Purification and characterization of hydroxycinnamoyl D-glucose. Quinate hydroxycinnamoyl transferase in the root of sweet potato, Ipomoea batatas Lam

We have previously proposed a chlorogenic acid biosynthetic pathway which involves a transesterification reaction between hydroxycinnamoyl D-glucose and D-quinic acid. The proposed pathway was based on tracer experimental results (Kojima, M., and Uritani, I. (1972) Plant Cell Physiol. 13, 311-319). The enzyme that catalyzes the above reaction has been purified 160-fold from sweet potato root (Ipomoea batatas Lam.) and characterized. The purified enzyme yielded one band of 26,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 25,000 by gel filtration chromatography. Therefore, the enzyme seems to consist of a single polypeptide of 25,000-26,000 daltons. The isoelectric point of the enzyme was 8.6. The optimum pH of the enzyme reaction was 6.0. The enzyme did not require any metal for activity and showed a broad substrate specificity toward hydroxycinnamoyl D-glucose as donors. The Km and Vmax values were 3.7 mM and 8.5 units/mg of protein for t-cinnamoyl D-glucose, 3.9 mM and 15.1 units/mg of protein for p-coumaroyl D-glucose, and 14.3 mM and 38.1 units/mg of protein for caffeoyl D-glucose. The enzyme showed a strict substrate specificity toward D-quinic acid-related compounds as acceptors; the Km and Vmax values were 16.7 mM and 15.1 units/mg of protein for D-quinic acid, 250 mM and 19.0 units/mg of protein for shikimic acid, and there was no activity with either L-malic acid or meso-tartaric acid. The enzyme activity changed in a manner suggesting its involvement in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.     

Ketopantoic acid reductase of Pseudomonas maltophilia 845. Purification, characterization, and role in pantothenate biosynthesis

Ketopantoic acid reductase (EC 1.1.1.169), an enzyme that catalyzes the formation of D-(-)-pantoic acid from ketopantoic acid, was purified 6,000-fold to apparent homogeneity with a 35% overall recovery from Pseudomonas maltophilia 845 and then crystallized. The relative molecular mass of the native enzyme, as estimated by the sedimentation equilibrium method, is 87,000 +/- 5,000, and the subunit molecular mass is 30,500. The enzyme shows high specificity for ketopantoic acid as a substrate (Km = 400 microM, Vm = 1,310 units/mg of protein) and NADPH as a coenzyme (Km = 31.8 microM). Only 2-keto-3-hydroxyisovalerate (Km = 8.55 mM, Vm = 35.8 units/mg) was reduced among a variety of other carbonyl compounds tested. The reaction is reversible (Km for D-(-)-pantoic acid = 52.1 mM), although the reaction equilibrium greatly favors the direction of D-(-)-pantoic acid formation. That the enzyme is responsible for the synthesis of D-(-)-pantoic acid necessary for the biosynthesis of pantothenic acid in P. maltophilia 845 is indicated by the observations that only this enzyme is missing in D-(-)-pantoate (or pantothenate)-requiring mutants derived from P. maltophilia 845 among several enzymes (i.e. ketopantoyl lactone reductase (EC 1.1.1.168) and acetohydroxy acid isomeroreductase (EC 1.1.1.86], which may be concerned in the formation of D-(-)-pantoic acid, assayed, whereas it is present in substantial amounts in the parent strain and in spontaneous revertants of the mutants.     

Isolation, crystallization, and properties of indolyl-3-alkane alpha-hydroxylase. A novel tryptophan-metabolizing enzyme.

Indolyl-3-alkane alpha-hydroxylase, a novel tryptophan-metabolizing enzyme, was prepared in crystalline form from soil isolate organism Pseudomonas XA. Emission spectroscopy and atomic absorption analyses of purified enzyme revealed the presence of iron (0.8 mol/mol of protein), and a number of observations supported the presence of heme prosthetic group (1.1 mol/mol of protein). The S20,w value of indolyl-3-alkane alpha-hydroxylase is 10.2 S, and the molecular weight by sedimentation equilibrium ultracentrifugation is 250,000. The E1%280 of the enzyme is 21, and the isoelectric point by isoelectric focusing on ampholine polyacrylamide gel plates is 4.8. The enzyme catalyzes hydroxylation on the side chain of a variety of 3-substituted indole compounds, including certain tryptophan-containing oligopeptides. The reaction product from tryptamine was identified by proton nuclear magnetic resonance and gas chromatography/mass spectroscopy analyses. While the indole ring remained intact, hydroxylation occurred at the side chain carbon adjacent to the ring. Nuclear magnetic resonance studies indicated that hydroxylation always took place at the same position when the substrate was tryptophan methyl ester, tryptophol, indole-3-propionate, or indole-3-butyrate. No other chemical change occurred when these substrates were incubated with the enzyme. The Km value of indolyl-3-alkane alpha-hydroxylase for L-tryptophan is 2.4 X 10(-6) M, at pH 7.2. The enzyme is inhibited by potassium cyanide (0.1 mM) or hydroxylamine (1mM), but not by NaBH4 (25 mM), aminooxyacetic acid (7mM), quinacrine (1 mM), chlortetracycline (1 mM), p-mercuribenzoate (0.1 mM), or ethylenediaminetetraacetate (1 mM). The plasma half-life (t1/2) of indolyl-3-alkane alpha-hydroxylase in tumor-bearing mice is approximately 25 h.     

Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase.

The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme that differs from other carbamoyl-phosphate synthetases and that resembles carbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomeric organization. This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhibit bicarbonatedependent ATPase activity. We have reexamined these findings using the enzyme of P. furiosus expressed in Escherichia coli from the corresponding gene cloned in a plasmid. We show that the enzyme uses chemically made carbamate rather than ammonia and bicarbonate and catalyzes a reaction with the stoichiometry and equilibrium that are typical for CK. Furthermore, the enzyme catalyzes actively full reversion of the CK reaction and exhibits little bicarbonate-dependent ATPase. In addition, it cross-reacts with antibodies raised against CK from Enterococcus faecium, and its three-dimensional structure, judged by x-ray crystallography of enzyme crystals, is very similar to that of CK. Thus, the enzyme is, in all respects other than its function in vivo, a CK. Because in other organisms the function of CK is to make ATP from ADP and CP derived from arginine catabolism, this is the first example of using CK for making rather than using CP. The reasons for this use and the adaptation of the enzyme to this new function are discussed.     

Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration

Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+).