M. Tsuchida

Abstract: Two different pathways for the bacterial oxidation of tryptophan have been proposed recently as a result of two independent investigations by the analysis of adaptive patterms. Suda, Hayaishi, and Oda (1949, 1950) used as the startingpoint for their work certain earlier observations by Mirick (1943) on the adaptive responses of an unidentified soil bacterium. Mirick's organism, which was potentially capable of oxidizing all three mono-aminobenzoic acids, could not be adapted to anthranilic (o-aminobenzoic) acid by exposure to either mor p-aminobenzoic acid; yet exposure to tryptophan, despite its very different chemical structure, caused an activation of the enzyme system oxidizing anthranilic acid equivalent to that produced by the specific substrate itself. The biochemical implication of these findings was comprehended by Suda, Hayaishi, and Oda, who formulated independently of others (Stanier, 1947; Karlsson and Barker, 1948) the concept that exposure to a primary substrate will activate adaptive enzymes which operate on the subsequent metabolic intermediates, and coined the name \"successive adaptation\" for the phenomenon. They proceeded to study the adaptive patterns caused by exposure to tryptophan of an unidentified Pseudomonas sp., deducing from their results the following metabolic sequence: L-tryptophan -> L-kynurenine -anthranilic acid -* catechol. Independently, Stanier and Tsuchida (1949) made a study of the oxidation of tryptophan by another unidentified pseudomonad, using the analysis of adaptive patterns. The general technique had been developed earlier by Stanier (1947) under the name of \"simultaneous adaptation\" during studies on the oxidation of simple aromatic compounds, and it had seemed of interest to find out whether other bacterial oxidations could be analyzed usefully by the same method. The results obtained with tryptophan were clear-cut and suggested as the initial degradative steps: tryptophan -kynurenine -kynurenic acid, anthranilic acid being excluded as a later intermediate. Both isomers of tryptophan could be oxidized. Thus two radically different pathways for the oxidation of tryptophan, diverging at the stage of kynurenine, have been reported as existing in two unidentified members of the genus Pseudomonas. Further work was clearly desirable, and we have consequently embarked on a comparative analysis of the

Abstract: Evans (1947) observed that cultures of Vibrio 01 developing at the expense of phenol gave an intense violet Rothera reaction during the later stages of growth. The Rothera test is a nitroprusside reaction used clinically to detect the presence of \"acetone bodies\" (acetone and acetoacetic acid) in urine. Although Evans did not succeed in identifying the substance responsible for the positive Rothera reaction in cultures of Vibrio 01, he established the fact that it was an organic acid that was considerably less ether-soluble than acetoacetic acid. Subsequently Kilby (1948) isolated the acid from culture filtrates of Vibrio 01 and identified it as ,3-ketoadipic acid, a 6-carbon dicarboxylic acid. He concluded that it was an intermediate in the oxidation of the benzene ring. As reported in an earlier paper (Sleeper, Tsuchida, and Stanier, 1950), we have succeeded in obtaining dried cell preparations of Pseudomonasfluorescens capable of oxidizing catechol and protocatechuic acid. The dried cell preparations were found to attack both substrates with a much lower oxygen uptake per mole than that which occurs during the equivalent oxidations by living cells, a fact which suggested that the oxidations by dried cells are blocked at an early stage. The investigations reported in the present paper show that both aromatic compounds are converted quantitatively by dried cells into ,B-ketoadipic acid, thus confirming in a direct manner Kilby's inference that this aliphatic acid is an intermediate in the oxidations of aromatic compounds.

Abstract: Our evidence concerning the pathways for the oxidation of aromatic compounds by Pseudomonas fluorescens (Stanier, 1947, 1948; Sleeper and Stanier, 1950) has been accumulated almost entirely by use of the technique of simultaneous adaptation. Although we believe that the logical basis of this method is sound, the technique is necessarily an indirect one. Consequently it appeared desirable to confirm the reactions postulated by isolation and study of the various enzymes involved. The present paper is concerned with preliminary phases of this work: the techniques for preparing dried cells and the effect of pre-established adaptive patterns on enzymatic activity in vitro.

Abstract: A survey of the pathways of tryptophan oxidation by pseudomonads (Stanier, Hayaishi, and Tsuchida, 1951) revealed the existence of two strains that attack tryptophan with a very low oxygen uptake per mole of substrate oxidized and are incapable of oxidizing at significant rates either kynurenic or anthranilic acid, key intermediates on the main alternate pathways for the oxidation of this compound. Data on the oxidative metabolism of tryptophan by these two strains are presented later. The methods employed have been previously described (Stanier, Hayaishi, and Tsuchida, 1951; Stanier and Hayaishi, 1951).

Abstract: Two new intermediates were identified in the protocatechuate pathway of Pseudomonas putida. The first of these, γ-carboxymuconolactone (γ-carboxy-γ-carboxymethyl-Δα-butenolide), is the product of the enzymic lactonization of β-carboxy-cis, cis-muconate. Enzymic decarboxylation of γ-carboxymuconolactone gives rise to β-ketoadipate enollactone (γ-carboxymethyl-Δβ-butenolide), the second newly discovered intermediate in the protocatechuate pathway. β-Ketoadipate enol-lactone, which was isolated and physically characterized, is also an intermediate in the catechol pathway; the catechol and protocatechuate pathways converge at this point. β-Ketoadipate enol-lactone is hydrolyzed to β-ketoadipate by an enzyme which is essential for utilization of either catechol or protocatechuate. Studies with Moraxella lwoffii showed that this organism also degrades protocatechuate and catechol by the pathways characteristic of P. putida. Elucidation of the bacterial pathway for the dissimilation of protocatechuate revealed that the three step-reactions responsible for the conversion of this compound to β-ketoadipate enol-lactone are analogous with the step-reactions responsible for the conversion of catechol to β-ketoadipate enol-lactone.

Abstract: Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91:1140–1154. 1966.—The control of synthesis of the five enzymes responsible for the conversion of d(−)-mandelate to benzoate by Pseudomonas putida was investigated. The first three compounds occurring in the pathway, d(−)-mandelate, l(+)-mandelate, and benzoylformate, are equipotent inducers of all five enzymes. A nonmetabolizable inducer, phenoxyacetate, also induces synthesis of these enzymes; but, unlike the metabolizable inducer-substrates, it does not elicit synthesis of enzymes that mediate steps in the pathway beyond benzoate. Under conditions of semigratuity, dl-mandelate elicits immediate synthesis at a steady rate of the first two enzymes of the pathway, but two enzymes which act below the level of benzoate are synthesized only after a considerable lag. Succinate and asparagine do not significantly repress the synthesis of the enzymes responsible for mandelate oxidation.

Abstract: The enzymes which catalyze reactions of molecular oxygen occur in three principle classes: (i) oxygen transferases, (ii) mixed function oxidases, and (iii) electron transferases. The first class catalyzes the transfer of a molecule of molecular oxygen to substrate. The second class catalyzes the transfer of one atom of the oxygen to substrate; the other atom undergoes two-equivalent reduction. The third class catalyses the reduction of molecular oxygen to hydrogen peroxide or to water.

Abstract: Publisher Summary β -ketoadipate pathway is considered one of the major microbial pathways for the dissimilation of aromatic and hydroaromatic compounds It provides a mechanism for the utilization of many different primary substrates, and offers a wide taxonomic distribution, both among bacteria and among fungi Various primary substrates dissimilated through this pathway are initially converted, through special reaction sequences, either to protocatechuic acid or to catechol These diphenols are regarded as the sites of entry to the two parallel and convergent branches of the β -ketoadipate pathway This chapter discusses the central reactions of the pathway—namely, those which result in the conversion of the two diphenolic intermediates to succinate and acetyl-CoA The most striking chemical feature of the bacterial β -ketoadipate pathway is the close chemical analogy between the successive metabolites of the catechol and protocatechuate branches, which differ only by the presence or absence of a carboxyl substituent The presence or absence of a carboxyl substituent on the substrates causes a major difference in chemical reactivity and imposes different requirements on the active sites of the enzymes operative in the two branches of the pathway