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.

The purification and properties of L-histidine--2-oxoglutarate aminotransferase from Pseudomonas testosteroni.

1. Inducible L-histidine--2-oxoglutarate aminotransferase was purified some 170-fold from extracts of Pseudomonas testosteroni. 2. The preparation showed only one major component after electrophoresis on polyacrylamide gels, though additional minor bands were observed when samples concentrated on a DEAE-cellulose column were used. 3. The molecular weight of the enzyme was found to be approx. 70000 by chromatography on Sephadex G-200. 4. The purification scheme produced enzyme that was inactive in the absence of pyridoxal 5'-phosphate. 5. The equilibrium constant for the reaction L-histidine+2-oxoglutarate equilibrium imidazolylpyruvate+L-glutamate was 0.49. 6. The reaction mechanism was Ping Pong. 7. The enzyme was shown to have only low activity towards aromatic amino acids and was highly specific for 2-oxoglutarate.

Amino acid sequence of a peptide containing the active cysteine residue of histidine ammonia-lyase.

1. Oxidized (polymerized) histidine ammonia-lyase from Pseudomonas testosteroni was activated with dithiothreitol and the reduced disulphide-linked cysteine residues of the native enzyme were carboxymethylated with iodo[(14)C]acetate. 2. The activity of the carboxymethylated enzyme was similar to that of the polymerized form and approx. 15% of that of the fully reduced form. 3. A tryptic digest of the [(14)C]carboxymethylated enzyme contained only one radioactive peptide. 4. The amino acid sequence of this peptide was shown to be Gly-Leu-Leu-Asp-Gly-Ser-Ala-Ile-Asn-Pro-Ser-His-Pro-Asn-Cys- (CH(2)CO(2)H)-Gly-Arg. 5. These findings show that, during polymerization, the disulphide bonds are formed between identical regions of the enzyme, and that the cysteine residue involved is also the one required in the reduced state for full activity of the enzyme.

The degradation of L-histidine in the rat. The formation of imidazolylpyruvate, imidazolyl-lactate and imidazolylpropionate.

1. Soluble and mitochondrial forms of histidine-pyruvate aminotransferase were separated from rat liver preparations by chromatography on DEAE-cellulose. 2. These enzymes were characterized with respect to substrate specificity, substrate affinity, pH optimum, stability and molecular weight by chromatography on Sephadex G-200. 3. Each enzyme has a relatively broad specificity showing significant activity towards l-phenylalanine and l-tyrosine and catalysing transamination with a number of monocarboxylic 2-oxo acids. 2-Oxoglutarate is not a substrate for either enzyme. 4. The molecular weights of the two enzymes, by chromatography on Sephadex G-200, are in the range 130000-150000. 5. The formation in vitro of imidazolyl-lactate from imidazolylpyruvate and NADH was demonstrated by using liver preparations. 6. From a study of imidazolyl-lactate-NAD(+) oxidoreductase activity after electrophoresis of liver preparations on polyacrylamide gel, and from an examination of the activity of l-lactate-NAD(+) oxidoreductase (EC 1.1.1.27) towards imidazolylpyruvate, it is concluded that this latter enzyme is responsible for the formation of imidazolyl-lactate in the liver. 7. Preparations of bacteria obtained from rat faeces form imidazolylpropionate from l-histidine and urocanate without further subculture. The amount of imidazolylpropionate formed is increased under anaerobic conditions and more so in an atmosphere of H(2). It is suggested that the gut flora of the rat contribute largely, if not exclusively, to the formation of imidazolylpropionate normally found in the urine.

The degradation of L-histidine, imidazolyl-L-lactate and imidazolylpropionate by Pseudomonas testosteroni.

1. Imidazol-5-ylpropionate and imidazol-5-yl-lactate are degraded by Pseudomonas testosteroni via inducible pathways. 2. Growth on either compound as the sole source of carbon results in the induction of the enzymes for histidine catabolism. 3. The pathway of histidine degradation in this organism, a non-fluorescent Pseudomonad, is shown to be the same as that operating in Pseudomonas fluorescens and Pseudomonas putida. It consists of the successive formation of urocanate, imidazol-4-on-5-ylpropionate, N-formimino-l-glutamate, N-formyl-l-glutamate and glutamate. 4. Whole cells of P. testosteroni accumulate urocanate in the reaction mixture when incubated with imidazolylpropionate, but only after an adaptive lag period which is removed by previous growth on imidazolylpropionate as the source of carbon. 5. Imidazolyl-lactate is oxidized to imidazolylpyruvate, which then gives rise to histidine by specific transamination with l-glutamate. 6. Cells grown on histidine, urocanate or imidazolylpropionate are also able to degrade imidazolyllactate. 7. Mutants lacking urocanase are unable to grow on imidazolylpropionate, imidazolyl-lactate, histidine or urocanate. One with impaired histidase activity cannot utilize histidine or imidazolyl-lactate, but grows normally on imidazolylpropionate or urocanate. A mutant unable to grow on imidazolylpropionate is indistinguishable from the wild-type with respect to growth on histidine, imidazolyl-lactate or urocanate. 8. Thus it is established that imidazolyl-lactate is metabolized via histidine whereas imidazolylpropionate enters the histidine degradation pathway after conversion into urocanate.

The control of the enzymes degrading histidine and related imidazolyl derivates in Pseudomonas testosteroni.

1. The induction of the enzymes for the degradation of l-histidine, imidazolylpropionate and imidazolyl-l-lactate in Pseudomonas testosteroni was investigated. 2. The activities of histidine ammonia-lyase, histidine-2-oxoglutarate aminotransferase and urocanase are consistent with these enzymes being subject to co-ordinate control under most growth conditions. However, a further regulatory mechanism may be superimposed for histidase alone under conditions where degradation of histidine must take place for growth to occur. 3. Experiments with a urocanase(-) mutant show that urocanate is an inducer for the enzymes given above and also for N-formiminoglutamate hydrolyase and N-formylglutamate hydrolase. 4. N-Formiminoglutamate hydrolase and N-formylglutamate hydrolase are also induced by their substrates, and it is suggested that these two enzymes may be different gene products from those expressed in the presence of urocanate. 5. Induction of the enzyme system for the oxidation of imidazolylpropionate is dependent on exposure of cells to this compound.

The metabolism of D-glucarate by Pseudomonas acidovorans.

1. Dehydratases that converted d-glucarate into 4-deoxy-5-oxoglucarate were partially purified from Klebsiella aerogenes and Pseudomonas acidovorans. 2. When d-glucarate was metabolized to 2,5-dioxovalerate it appeared that water and carbon dioxide were removed from 4-deoxy-5-oxoglucarate in one enzymic step: 4,5-dihydroxy-2-oxovalerate was not an intermediate in this reaction. 3. A method for the enzymic determination of d-glucarate is described.

Purification and properties of D-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating).

1. An enzyme extracted from Pseudomonas acidovorans was purified and shown to catalyse the simultaneous dehydration and decarboxylation of d-4-deoxy-5-oxoglucarate. It is proposed to name the enzyme d-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating), trivial name ;deoxyoxoglucarate dehydratase'. 2. No added cofactors were required, and the enzyme was inactivated when incubated with its substrate in the presence of sodium borohydride. Under these conditions the substrate and enzyme appeared to be bound covalently. 3. The action of the enzyme is readily explained if it is assumed that d-4-deoxy-5-oxoglucarate forms a Schiff base with a lysine residue in the enzyme.