Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida.

Urocanase is inactivated in intact cells of Pseudomonas putida and photoactivated by brief exposure of the cells to the UV radiation in sunlight. The dark reversion (inactivation) in vitro is explained by the formation of a sulfite-NAD adduct. Our objective was to investigate the dark reversion in vivo. Various compounds were added to P. putida cells, and the reversion was measured, after sonication, by comparison of the activity before and after UV irradiation. Sulfite, cysteine sulfinate, and hypotaurine enhanced the reversion of urocanase in resting cells. The reversion was time and concentration dependent. Sulfite modified the purified enzyme, but cysteine sulfinate and hypotaurine could not, indicating that those two substances had to be metabolized to support the reversion. Both of those compounds yielded sulfite when they were incubated with cells. Transaminases form sulfite from cysteine sulfinate. P. putida extract contained a transaminase whose activity involved as alpha-keto acid and either cysteine sulfinate or hypotaurine for (i) production of sulfite, (ii) disappearance of substrates, (iii) formation of corresponding amino acids, and (iv) urocanase reversion. Porcine crystalline transaminase caused reversion of highly purified P. putida urocanase with cysteine sulfinate and alpha-ketoglutarate. We conclude that in P. putida cysteine sulfinate or hypotaurine is catabolized in vivo by a transaminase reaction to sulfite, which modifies urocanase to a form that can be photoactivated. We suggest that this photoregulatory process is natural because it occurs in cells with the aid of sunlight and cellular metabolism.     

The purification and properties of urocanase from Pseudomonas testosteroni.

Urocanase (urocanate hydratase, EC 4.2.1.49) purified from Pseudomonas testosteroni has a mol.wt. of 118000 determined by sedimentation-equilibrium analysis. Ultracentrifugation in 6M-guanidine hydrochloride and polyacrylamide-gel electrophoresis in sodium dodecyl sulphate show that the enzyme consists of two identical or very similar subunits. It is, like urocanase isolated from other sources, inhibited by reagents that react with carbonyl groups. Although urocanase from Ps. testosteroni is strongly inhibited by NaBH4, no evidence could be obtained for the presence of covalently bound 2-oxobutyrate as a prosthetic group; this is in contrast with findings elsewhere for urocanase from Pseudomonas putida. Urocanase from Ps. testosteroni does not contain pyridoxal 5'-phosphate as a coenzyme and in this respect is similar to all urocanases studied in purified form.     

Substrate-mediated inactivation of urocanase from Pseudomonas putida. Evidence for an essential sulfhydryl group

Incubation of urocanase from Pseudomonas putida with either its substrate, urocanic acid, or product, 4'(5')-imidazolone-5'(4')-propionic acid, resulted in an oxygen-dependent inhibition of enzyme activity. Coincident with the inactivation was the stoichiometric incorporation of radioactivity from [14C]urocanate into the protein. NAD+ which is required for activity or urocanase was not directly involved in the inactivation process. The inactivation of urocanase was irreversible, could be partially blocked by the competitive inhibitor imidazolepropionate, and involved the modification of a single active-site thiol. The inhibition resulted from oxidative decomposition of 4'(5')-imidazolone-5'(4')-propionate but was not due to the formation of the major degradative product, 4-ketoglutaramate, since this compound was not an irreversible inactivator of urocanase although it did produce some inhibition at high concentrations. A mechanism is presented in which a reactive imine intermediate in the decomposition scheme is subject to nucleophilic attack by an active-site thiol, thereby generating a covalent enzyme--thioaminal adduct. These results emphasize the importance of a catalytic center sulfhydryl group for urocanase activity.     

The stoichiometry of the tightly bound NAD+ in urocanase. Separation and characterization of fully active and inhibited forms of the enzyme

1. Urocanase, purified by classical methods [Keul, V., Kaeppeli, F., Ghosh, C., Krebs, T., Robinson, J. A. and Rétey, J. (1979) J. Biol. Chem. 254, 843-851] from Pseudomonas putida was submitted to high-performance liquid chromatography on a TSK-DEAE column. The enzyme was eluted in three resolved peaks (A, B and C) exhibiting specific activities of 3.4 U/mg, 1.85 U/mg and 0.4 U/mg, respectively. 2. The difference spectra of peaks B and A as well as of C and A showed maxima at 330 nm. 3. Irradiation of peaks B and C at 320 nm resulted in an increase of urocanase activity by 45% and 400%, respectively. Peak A could not be photoactivated. Rechromatography of the photoactivated peaks B and C on the TSK-DEAE column confirmed their partial transformation into peak A. 4. Spectroscopic methods for quantitative protein determination were adapted to urocanase. The stoichiometry of bound NAD+/urocanase (form A) was determined to be 1.75 by enzymic analysis of the free NAD+ released upon acid denaturation of the holoenzyme. A similar stoichiometry (1.8-1.9) was found for all three forms (A, B and C) by biosynthetic incorporation of [7-14C]nicotinate into urocanase using a nicotinate auxotrophic mutant of P. putida. 5. Form A of urocanase showed, after treatment with NaBH4 up to 50% inhibition, an elution pattern (TSK-DEAE column) similar to a mixture of forms A, B and C in the approximate ratio of 1:2:1. None of these forms could be photoactivated. 6. We conclude that form A of the urocanase dimer contains two intact NAD+ molecules. In form B one of the two subunits contains an NAD+-nucleophile adduct which is present in both subunits of form C. Full urocanase activity requires intact NAD+ in both subunits. Intact NAD+ can be regenerated from the adduct but not from the reduced form by photolysis. The two subunits of urocanase are independent both in their catalytic activity and in modification reactions.     

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.     

Involvement of Threonine Dehydratase in Biosynthesis of the α-Ketobutyrate Prosthetic Group of Urocanase

Seventeen mutants of Pseudomonas putida that were unable to grow on threonine as nitrogen source owing to a lack of threonine dehydratase were isolated, and all were found to be unable to synthesize active urocanase. Spontaneous revertants selected for urocanase production concomitantly regained threonine dehydratase. Mutants that were unable to utilize urocanate as carbon source were also isolated, and these were defective in urocanase formation but were normal in threonine dehydratase levels. Since alpha-ketobutyrate is the prosthetic group for urocanase, these results are consistent with the proposal that threonine dehydratase is necessary for urocanase prosthetic group biosynthesis. However, the lack of urocanase activity in threonine dehydratase-negative mutants was shown not to be the result of reduced levels of endogenous free alpha-ketobutyrate, nor to the participation of threonine dehydratase in the initiation of urocanase biosynthesis through the conversion of threonyl-tRNA(Thr) to alpha-ketobutyryl-tRNA(Thr). Other alternatives for the participation of threonine dehydratase in urocanase biosynthesis are discussed.     

GABA from putrescine is bound in macromolecular form in keratinocytes

We report for the first time that the neurotransmitter γ-aminobutyric acid (GABA) exists in macromolecular form in keratinocytes. GABA derived from putrescine (Pu) has been identified as a component of acid-precipitable material of cultured human keratinocytes. Confluent, stratified cultures of human foreskin keratinocytes exposed to [³H]-Pu for 4 hours took up about 14% of the radioactivity from the medium and 1% of the total cell-associated radioactivity was precipitable by trichloroacetic acid (TCA). Both attached and shed cells were examined by HPLC for Pu and its radioactive metabolites in TCA-insoluble and TCA-soluble fractions. GABA accounted for the major portion (54%) of the radioactivity derived from Pu in the TCA-precipitable material of attached keratinocytes. Pu and spermidine represented lesser amounts, 35% and 9% respectively, of the total TCA-precipitable radioactivity. In addition, a large portion of acid soluble radioactivity derived from Pu (63%) was GABA, whereas Pu and spermidine represented 29% and 6% respectively of the total TCA-soluble radioactivity. The exact origin of GABA in acid-precipitable material, as well as its form of attachment, is currently under investigation.     

Mechanism of action of urocanase. Specific 13C-labelling of the prosthetic NAD+ and revision of the structure of its adduct with imidazolylpropionate

1. [4-13C]Nicotinate was synthesised and used to support the growth of a nicotinate auxotrophic mutant of Pseudomonas putida. 13C-NMR spectroscopy of the isolated urocanase confirmed the efficient incorporation of 13C into C4 of the nicotinamide ring of the tightly bound NAD+ cofactor. 2. beta-[( 2'-13C]Imidazol-4-yl)propionate was synthesised according to known procedures and used for inhibition of the 13C-labelled urocanase. An increase in the absorbance at 330 nm indicated adduct formation between enzyme-bound NAD+ and inhibitor. The adduct was stabilised by oxidation with phenazine methosulfate and isolated using a slight modification of the procedure of Matherly et al. [Matherly, L. H., DeBrosse, C. W. & Phillips, A. T. (1982) Biochemistry 21, 2789-2794]. 3. The 13C-NMR spectrum of the doubly labelled adduct, [4-13C]NAD-[2'-13C]imidazolylpropionate, showed no one-bond 13C-13C coupling between labelled sites. The 1H-NMR spectrum of this adduct in 2H2O showed only one imidazole signal, which appeared as a doublet (1JC-H = 212 Hz), confirming the presence of a proton at the labelled C2'. The lack of a C5' signal and further NMR data provide evidence for a C-C bond between C4 of the nicotinamide and C5' of the imidazole ring. 4. The revised structure for the enzymatically formed addition complex suggests a novel mechanism for the urocanase reaction which is not only chemically plausible but also explains the previously observed urocanase-catalysed exchange of the C5 proton of urocanate and of beta-(imidazol-4-yl)propionate.     

Activation of urocanase from Pseudomonas putida by electronically excited triplet species

Urocanase from Pseudomonas putida becomes inactive in growing and resting cells and, as shown previously, is activated by the direct absorption of ultraviolet light. In this study, we describe the activation of urocanase by energy transfer from triplet indole-3-aldehyde, generated in the peroxidase-catalyzed aerobic oxidation of indole-3-acetic acid. The activation was time-, temperature-, and pH-dependent. The involvement of reactive oxygen intermediates was excluded by the lack of effect of appropriate quenchers and traps. Triplet quenchers, in contrast, reduced the level of activation. Photoexcited rose bengal, a triplet species of a different nature and origin, was also effective in promoting activation. These results demonstrate a potential mechanism of urocanase regulation not dependent on an environmental source of light, but rather brought about by an enzymically generated excited species.     

Studies on lysophospholipases: VII. Synthesis of acylthioester analogs of lysolecithin and their use in a continuous spectrophotometric assay for lysophospholipases,a method with potential applicability to other lipolytic enzymes

The synthesis of acylthioester analogs of lysolecithins, i.e., 2-hexadecanoylthio-1-ethyl-phosphorylcholine and 3-hexadecanoylthio-1-propyl-phosphorylcholine is described. Both compounds were found to be hydrolysed by a homogeneous lysophospholipase from beef liver, a spectrophotometric assay for the activity of which was developed by continuous measurement of the released thiol groups in the presence of dithionitrobenzoic acid.Phospholipase A₂ from pig pancreas effected hydrolysis of the acylthioester bond in 2-hexadecanoylthio-1-ethyl-phosphorylcholine, the enzymatic action of which could also be monitored spectrophotometrically.Lipase from pig pancreas was found to hydrolyse acylthioester bonds in 2-hexadecanoylthio-1-ethanol. The tributyryl ester of 3-mercapto-1,2-propanediol was synthesized and used to compare the release of total acid and thiol groups during hydrolysis with lipase. A ratio of about 2:1 was found for these releases.These findings clearly indicate the potential applicability of acylthioester analogs of substrates for phospholipases, lysophospholipases, and lipases in continuous spectrophotometric assays for lipolytic enzymes.     

恶臭假单胞菌NA-1菌株烟酸羟基化酶活性的诱导和转化条件的研究

恶臭假单胞菌NA-1菌株的培养和产酶特性与已报道的产酶菌株粘质沙雷氏菌(Serratiamarcescens)IFO12648和荧光假单胞菌(Psudomonasfluorescens)TN5有所不同,主要反映在最适碳源及浓度、最适诱导剂浓度和最适培养温度等方面。最适的转化条件是温度为30℃,pH为7.0,烟酸的浓度为3%。采用初步优化后的条件和流加底物的方式进行4L上罐生产,恶臭假单胞菌NA-1菌株的6-羟基烟酸产率可达到108.39gL。     

Identification of the enol tautomer of imidazolone propionate as the urocanase reaction product

A fluorescent product was transiently formed during catalysis by urocanase from Pseudomonas putida. The fluorophore showed an emission maximum at 430 nm when excited at 330 nm, essentially identical to that exhibited by the enol tautomer of imidazolone propionate. The keto isomer was not fluorescent under these conditions. In aqueous acid solutions where imidazolone propionate is relatively stable, an equilibrium mixture of tautomeric forms contained approximately 1% of the enol isomer. In ethanolic solutions, the equilibrium concentration of enol tautomer increased to approximately 25%. The differing content of imidazolone propionate tautomers as a function of solvent conditions permitted a comparison of the keto and enol forms as substrates for the reverse reaction. This revealed an almost complete preference for the enol tautomer. These results are taken as direct proof that enol imidazolone propionate is the true urocanase reaction product.     

Regulation of histidine catabolism by succinate in Pseudomonas putida

The regulation of the histidine-degrading pathway is known to involve induction and repression. Our studies have shown that succinate may control the histidine-degrading pathway by sequential negative feedback inhibition. Succinate inhibited urocanase, and urocanate in turn inhibited histidase. Crude preparations of the two enzymes were made from Pseudomonas putida grown on l-histidine. Succinate was a competitive inhibitor of urocanase (K(i), 1.8 mm). Lactate, pyruvate, alpha-ketoglutarate, and glutamate did not inhibit urocanase. Urocanate inhibited histidase competitively (K(i), 0.13 mm). A multienzyme system (histidine to glutamate), when incubated with histidine and succinate, exhibited the combined effect. Succinate caused the level of accumulated urocanate to increase and indirectly blocked histidine disappearance. Growth of cells on urocanate as a nitrogen source was inhibited by 1% succinate. Succinate may play a physiological role in the biological regulation of histidine metabolism.     

一株烟酸羟基化转化菌株的筛选和鉴定

从南京地区的土壤中筛选到一株高效转化烟酸为 6_羟基烟酸的菌株NA_1。形态及生理生化特征测定结果表明 ,NA_1菌株与假单胞菌属 (Pseudomonas)中的恶臭假单胞菌 (P .putida)种的特征基本一致。测定了该菌株的16SrDNA序列并根据 16SrDNA构建了系统发育树 ;在系统发育树中 ,NA_1菌株与恶臭假单胞菌形成一个类群 ,序列同源性为 99%。因此将NA_1菌株鉴定为恶臭假单胞菌     

Specificity of sialytransferase: structure of alpha1-acid glycoprotein sialylated in vitro. A new methodology for the determination of the structure of the linkage formed by glycosyltransferase action on galactosyl-oligosaccharide-protein acceptors.

The terminal galactosyl units of desialylated alpha1-acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H-labeled glycoprotein was effective as an acceptor in sialytransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP-N-acetyl-neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H-labeled glycoprotein yielded radioactive 2,3,4-trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C-6 of the terminal galactosyl units of desialylated alpha1-acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions. This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native alpha1-acid glycoprotein.     

恶臭假单胞菌NA-1菌体培养转化和静息细胞转化联合工艺生产6-羟基烟酸研究

现有微生物羟基化烟酸采用的是静息细胞转化工艺。但研究揭示,恶臭假单胞菌NA-1(Pseudomonas putidaNA-1)在培养过程中不降解发酵液中由诱导剂烟酸转化形成的6-羟基烟酸,这是由于烟酸的存在抑制了羟基烟酸降解酶的作用,而不是因为细胞停止生长不利用羟基烟酸的缘故。因而尝试利用菌体诱导培养过程进行烟酸转化生产,建立了一种新的生产工艺,即菌体培养转化和静息细胞转化联合工艺。该工艺在恶臭假单胞菌NA-1培养过程中持续补充烟酸以维持1%(W/V)浓度,使烟酸被生长细胞转化为羟基化烟酸并在发酵液中线性积累,而不被进一步降解;培养转化结束后,发酵液中的静息细胞依然拥有很高的羟基化酶活力,能够再次用于转化反应。该联合转化工艺与传统的静息细胞转化工艺相比,不仅节约了诱导剂烟酸,而且6-羟基烟酸的产量提高了65%。     

Distribution in tissue homogenates,and the nature of the linkage of injected proteins to subcellular particles

Intravenously injected labeled proteins were recovered mostly in particulate fractions of rat liver homogenate. Distribution showed changes depending on the time elapsed from the injection. ¹³¹I-albumin undergoes an intraparticulate hydrolysis which shows the highest activity in the gradient fractions associated with the highest level of acid phosphatase. The labeled albumin-bearing particles separated at 27,000 g × 10 minutes released their radioactive protein at the same rate as acid phosphatase appeared in the medium, under the effect of such agents as distilled water, salts, homogenization, sonication and pH changes. The substitution of sucrose for distilled water or salts showed that the particles behave as an osmotic system as do lysosomes. These experiments prove that secondary lysosomes involved in the hydrolysis of foreign proteins, whose existence was shown by other authors only at the histochemical level, may survive the distrupting action of conventional homogenization and maintain many properties characteristic of primary lysosomes in addition to the ability of hydrolysing “in vitro” the engulfed material.     

Thioglycolate, competitive inhibitor of urocanase.

Urocanase was inhibited by thioglycolate, 2-mercaptoethanol, dithioerythritol, and 3-mercaptopropionate. Thioglycolate inhibited competitively at low concentrations (K_i, 0.1 mM) and protected the active site from modification by sulfite. The inhibited enzyme was reactivated by dialysis. A difference spectrum peak of 328 nm for the thioglycolate-urocanase complex compared to the 327 nm absorption maximum of the NAD-thioglycolate adduct. Several nucleophiles are known to inhibit urocanase. We conclude that thioglycolate, as a nucleophilic agent, inhibits by forming an adduct with the tightly bound NAD of urocanase. These results provide indirect evidence that NAD may be the locus of substrate binding in urocanase.