Isolation, sequencing and expression in E. coli of the urocanase gene from white clover (Trifolium repens).

The urocanase gene was detected in a clone obtained from a genomic library of white clover. The entire gene has been sequenced and expressed in the pT7-7/E. coli BL 21 (DE 3) system. The deduced sequence of the plant urocanase is 72% homologous with that of the well-characterized urocanase from Pseudomonas putida. The purification procedure, as well as kinetic and electrophoretic behaviour, of the new enzyme are described.

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.

Specific inhibition of bacterial and bovine urocanases by glycylglycine.

Urocanase (EC 4.2.1.49) purified from Pseudomonas putida was unexpectedly inhibited by the dipeptide glycylglycine. Using a spectrophotometric assay for urocanase activity, we characterized the inhibition. The inhibition was temperature-, concentration-, and time-dependent; 0.1, 0.5 and 1.0 mM glycylglycine inhibited the enzyme by 20%, 50% and 78%, respectively, in 60 min at 30 degrees C. Dithiothreitol and reduced glutathione did not prevent the process. The inhibition was a pseudo first-order reaction. Three ligands that bind to the active site, urocanate, imidazole-propionate (a competitive inhibitor) and sulfite, protected the enzyme from glycylglycine inhibition. The inhibition was very specific for glycylglycine, because fifteen related biochemicals, including glycine, triglycine, and tetraglycine, were not effective. Ethylenediaminetetraacetic acid and other chelators did not inhibit urocanase. Bovine liver urocanase was also inhibited by this peptide. The characteristics of this inhibition suggest that glycylglycine acts at the active site, does not function by metal binding and that minor alterations in the glycylglycine molecule preclude the inhibition. A specific inhibition of urocanases by glycylglycine has been observed.

[Purification, properties and regulation of urocaninase from rat liver]. / Ochistka, svoistva i reguliatsiia urokaninazy pecheni krys.

Urocaninase (EC 4.2.1.4.9) from rat liver homogenate has been purified, using protein precipitation at pH 4,8, ammonium sulfate fractionation, gel-filtration through Sephadex G-200 and chromatography on DEAE-cellulose. Upon DEAE-cellulose chromatography urocaninase is separated from the proteins possessing the activity of 3',5'-AMP-dependent protein kinase. The purified enzyme becomes activated after addition of ATP and exogenous protein kinase or one of the fractions resulting from DEAE-cellulose chromatography. Using [gamma-32P]ATP, it has been shown that such activation is accompanied by incorporation of at least one phosphate residue into the enzyme molecule. The mol. weight of urocaninase as determined by gel-filtration is about 110 000. The Km value for urocanate is 15 . 10(-6) M, the isoelectric point lies at 5,6. The mechanism of regulation of the urocaninase activity in rat liver is discussed.

Identification of the prosthetic group of urocanase. The mode of its reaction with sodium borohydride and of its photochemical reactivation.

Urocanase from Pseudomonas putida and from beef liver were isolated by modifying described procedures. Both enzymes were inactivated and labeled on treatment with tritiated sodium borohydride and gave, upon subsequent hydrolysis, a radioactive acid. The previously reported identity of this acid as 2-hydroxybutanoic acid was disproved by several criteria. Other hydroxy acids were also proved to be different from the radioactive acid derived from urocanase. A large portion of the radioactive material from P. putida was found to be nicotinic acid by 1H NMR spectroscopy, gas-liquid chromatography of its methyl ester, and co-crystallization with authentic reference compounds both as the acid and as the hydrazide. A significant portion of the radioactive material derived from beef liver urocanase also co-crystallized with nicotinic acid. Sodium borohydride-treated inactive urocanase was partially reactivated by light. The action spectrum of the photoreactivation showed a maximum at 330 nm. Treatment of urocanase with sodium borodeuteride followed by hydrolysis afforded a sample of nicotinic acid which carried deuterium mainly in position 6. Both the reversible reducibility of urocanase and its action spectrum of photoreactivation suggest that urocanase contains an enzyme-bound nicotinamide nucleotide molecule which is essential for enzymic activity.

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.

Catalytic and thermodynamic properties of the urocanate hydratase reaction.

Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.