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An enzyme of Alcaligenes eutrophus JMP 134 which catalyzes dechlorination of (4R, 5R)-and (4R, 5S)-5-chloro-3-methyl-and (4R, 5S)-5-chloromuconolactone to principally 3-methyl-trans-, 3-methyl-cis -dienelactone and cis -dienelactone, respectively, was purified to homogeneity. The enzyme was identified as muconolactone isomerase on the basis of its high activity with muconolactone and on its high degree of sequence similarity with previously described muconolactone isomerases. Molecular mass determinations of the highly hydrophobic and heat-resistant enzyme indicated a decameric structure involving a single 10.100-kDa subunit similar to that of muconolactone isomerase of Pseudomonas putida. Kinetic analysis showed cooperative effects between the subunits during conversion of (4R, 5S)-5-chloro-3-methylmuconolactone. (4R, 5S)-5-chloromuconolactone was the preferred substrate, over the natural substrate (4S)-muconolactone. The (4S, 5S)-structure was found to be an inhibitor of (4R, 5R)-5-chloro-3-methylmuconolactone transformation. Methylsubstitution of the substrate results in a higher affinity for the enzyme, but a drastically lower velocity, resulting in a lower specificity constant.

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl-CoA. The paa operon of E. coli encodes 14 polypeptides involved in the catabolism of phenylacetate. We have found that E. coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source. Incubation of a paaG mutant with [U-13C8]phenylacetate yielded ring-1,2-dihydroxy-1,2-dihydrophenylacetyl lactone as shown by NMR spectroscopy. Incubation of the paaF and paaH mutants with phenylacetate yielded delta3-dehydroadipate and 3-hydroxyadipate, respectively. The origin of the carbon atoms of these C6 compounds from the aromatic ring was shown using [ring-13C6]phenylacetate. The paaG and paaZ mutants also converted phenylacetate into ortho-hydroxyphenylacetate, which was previously identified as a dead end product of phenylacetate catabolism. These data, in conjunction with protein sequence data, suggest a novel catabolic pathway via CoA thioesters. According to this, phenylacetyl-CoA is attacked by a ring-oxygenase/reductase (PaaABCDE proteins), generating a hydroxylated and reduced derivative of phenylacetyl-CoA, which is not re-oxidized to a dihydroxylated aromatic intermediate, as in other known aromatic pathways. Rather, it is proposed that this nonaromatic intermediate CoA ester is further metabolized in a complex reaction sequence comprising enoyl-CoA isomerization/hydration, nonoxygenolytic ring opening, and dehydrogenation catalyzed by the PaaG and PaaZ proteins. The subsequent beta-oxidation-type degradation of the resulting CoA dicarboxylate via beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA appears to be catalyzed by the PaaJ, PaaF and PaaH proteins.

This work provides presumptive evidnce that Pseudomonas fluorescens metabolizes the higher homologs (C6 to C10) of the saturated dicarboxylic acid series by β-oxidation, mediated by a specific series of inducible enzymes. The initial step in the metabolism of four of these acids (pimelic, suberic, azelaic and sebacic acids) is the formation of the corresponding CoA-ester. This step is mediated by a single, inducible thiokinase. A thiokinaseless mutant can utilize none of these four dicarboxylic acids. Although adipic acid can also apparently be activated to a slight extent by the thiokinase, the unimpaired ability of the thiokinaseless mutant to grow with adipate shows that there is a second mechanism for the activation of this member of the series. Adipic acid is also activated by an inducible adipate: succinyl-CoA transferase, which cannot use the higher homologs of the dicarboxylic acid series as CoA acceptors (with the exception of pimelic acid, which is weakly activated). This transferase is probably responsible for adipic acid activation in vivo. Inducible dehydrogenases which can oxidise acyl-CoA and β-hydroxyacyl-CoA derivatives of the higher dicarboxylic acids were also demonstrated, but their substrate specificities were not determined. The former enzyme can dehydrogenate both adipyl-CoA and azelayl-CoA; and the latter, β-hydroxyadipyl-CoA. A mutant unable to form the enzyme with β-hydroxy-adipyl-CoA dehydrogenase activity cannot grow with any dicarboxylic acid of the C6 to C10 series.

1 Glucarate hydro-lyase, obtained from cells of Pseudomonas A grown on glucarate as the sole source of carbon, was purified 40-fold in 70% yield. 2 The effects of a variety of chemical modifiers and competitive inhibitors on the enzyme activity were studied. 3 Enzyme activity was irreversibly lost by incubating the enzyme with p-chloromercuribenzene sulphonate. 4 (+)-Tartrate, a competitive inhibitor of the enzyme, protected the enzyme from inhibition by this reagent but was not effective when the enzyme was inhibited by coupling it to a diazonium salt. Glucarate, unlike (+)-tartrate, was an effective protecting group against this type of inhibition. 5 The roles of the modified amino acid residues are discussed.

1 Pseudomonas arvilla mt-2 grows at the expense of benzoate, m-toluate (3-methylbenzoate) and p-toluate (4-methylbenzoate), but not o-toluate (2-methylbenzoate). Under various conditions compounds with spectra characteristic of muconic semialdehydes can be made to accumulate in the media. These spectra indicate that benzoate is metabolised through catechol and 2-hydroxymuconic semialdehyde (2-hydroxy-6-oxohexa-2,4-dienoate), m-toluate through 3-methylcatechol and 2-hydroxy-6-oxohepta-2,4-dienoate and p-toluate through 4-methylcatechol and 2-hydroxy-5-methylmuconic semialdehyde (2-hydroxy-5-methyl-6-oxohexa-2,4-dienoate). 2 Freshly harvested cells grown on all three substrates only consume oxygen at a significant rate when presented with the growth substrates themselves and catechol and the methylcatechols, but not with salicylate, protocatechuate or phenol or the cresols. 3 Extracts of cells grown on these substrates contain high induced levels of the suite of meta cleavage enzymes, including both the hydrolytic branch and the 4-oxalocrotonate branch. 4 The substrate specificity of the early enzymes of the pathway suggests that only one non specific enzyme expresses each activity in cell-free extracts and that it is nonspecifically induced during growth on all three carbon sources. 5 It is suggested that the metabolic role of the hydrolytic branch of the pathway is the assimilation of 3-methylcatechol and its precursor, and that of the 4-oxalocrotonate branch is to assimilate catechol and 4-methylcatechol and their precursors. 6 o-Toluate is not metabolised because it is neither a substrate for the benzoate oxidase system nor an inducer of any of the meta pathway enzymes. 7 It appears that benzoate and m-and p--toluates are the substrate inducers of the meta pathway enzymes in this organism. The ortho pathway is induced on incubating cells with catechol and the first inducer appears to be cis,cis-muconate or possibly catechol itself.

This work shows that Pseudomonas fluorescens can synthesize two different inducible enzymes which possess β-ketoadipate: succinyl-CoA transferase activity. Transferase I is operative in the activation of free β-ketoadipic acid and is specifically induced in cells grown either with this acid, or with an aromatic precursor (benzoate) oxidized through the β-ketoadipate pathway. Transferase II is specifically induced in cells grown with saturated dicarboxylic acids of chain length C6 through C10, and its physiological function is that of an adipate: succinyl-CoA transferase; its ability to activate β-ketoadipic acid reflects non-specificity with regard to the CoA acceptor.

The inducible enzymes responsible for the dissimilation of aromatic compounds through the β-ketoadipate pathway by Moraxella calcoacetica are subject to a high degree of coordinate control. The five enzymes which catalyze the conversion of protocatechuate to β-ketoadipyl-CoA are coordinately synthesized, the probable inducer being protocatechuate. In the conversion of catechol to β-ketoadipyl-CoA, there are two separate inductive events, though both are mediated by one metabolite-inducer, cis,cis-muconate. One is the induction of catechol oxygenase; the other is the coordinate induction of the four enzymes that catalyze conversion of cic,cis-muconate to β-ketoadipyl-CoA.

Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD-glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2- and HCO-3. The concentrations of L-aspartate and 2-oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl-CoA. Acetyl-CoA increases the sigmoidal character of the relationship: initial rate/[L-aspartate], but decreases this parameter for the relationship: initial rate/[2-oxoadipate]. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.

1 The ability to oxidize quinate is elicited in Acinetobacter calco-aceticus not only by growth on quinate but also by growth on protocatechuate or its precursors such as shikimate and p-hydroxybenzoate. Accordingly the synthesis of a quinate dependent dehydrogenase was found to be induced by protocatechuate. Moreover, this enzyme seems to belong to the same coordinate block of enzymes responsible for the conversion of shikimate to β-ketoadipyl-CoA. 2 Analogies between quinate and shikimate oxidation in A. calco-aceticus have been substantiated by showing that both quinate dehydrogenase and shikimate dehydrogenase activities rely on a single enzyme. 3 Several auxotrophs that have lost the ability to utilize both quinate and shikimate as sole source of carbon and energy have been isolated. Particularly relevant for this study are two of these mutants in which the dehydrogenase is severely impaired and correspondingly their ability to oxidize the mentioned hydroaromatic compounds is substantially reduced. The properties of these auxotrophs and their revertants confirm the key role of the dehydrogenase in the dissimilation of both quinate and shikimate. The rationale of the control of this enzyme by protocatechuate is discussed.

A benzene-utilizing strain of Moraxella B (genus/Acinetobacter) was isolated from Rhine water. Cell-free extracts of this bacterium, which is highly specific for the oxidation of benzene, require for this process catalytic amounts of NADH. This is regenerated during the course of the reaction by the dehydrogenation of cis-cyclohexadiene-diol to pyrocatechol. The oxygenase system contains nonheme-iron in the form of strongly bound Fe2+. It incorporates two atoms of oxygen simultaneously, as proven by isotope experiments; it is thus a dioxygenase. cis-1,2-Dihydroxy-dihydrobenzene was isolated as the first identifiable stable intermediate. This is further oxidized to pyrocatechol which is then eventually completely metabolized via the 3-oxoadipic acid pathway.