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A novel Fe + Zn containing oxygenase from Acinetobacter johnsonii catalyses 2,3-cleavage of acetylacetone to acetate and methylglyoxal has been purified. The stoichiometry of reactants and products conforms to a classical dioxygenase. The pure protein is a homotetramer of 64 kD with variable amounts of Fe 2+ and Zn 2+ . Activity of the enzyme is more closely related to the Fe 2+ content than to the amount of protein. A purification of acetylacetone 2,3-oxygenase, some of its physical properties, and the preference for some analogous substrates are described.

A revised purification of acetopyruvate hydrolase from orcinol-grownPseudomonas putidaORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of ∼38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by1H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three1H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A1H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized π-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg2+bound to the enzyme was made.1H–2H exchange reactions showed the complete, fast equilibration of2H into the C-3 of acetopyruvate chemically; this accounts for the appearance of2H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional1H-NMR in normal1H2O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe2+ is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a Km of 9.1μM and a kcat of 8.5s-1. A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.

Burkholderia strain (JT 1500), able to use 2-naphthoate as the sole source of carbon, was isolated from soil. On the basis of growth characteristics, oxygen uptake experiments, enzyme assays, and detection of intermediates, a degradation pathway of 2-naphthoate is proposed. The features of this pathway are convergent with those for phenanthrene. We propose a pathway for the conversion of 2-naphthoate to 1 mol (each) of pyruvate, succinate, and acetyl coenzyme A and 2 mol of CO2. During growth in the presence of 2-naphthoate, six metabolites were detected by thin-layer chromatography, high-performance liquid chromatography, and spectroscopy. 1-Hydroxy-2-naphthoate accumulated in the culture broth during growth on 2-naphthoate. Also, the formation of 2'-carboxybenzalpyruvate, phthalaldehydate, phthalate, protocatechuate, and beta-carboxy-cis,cis-muconic acid was demonstrated. (1R,2S)-cis-1,2-Dihydro-1,2-dihydroxy-2-naphthoate was thus considered an intermediate between 2-naphthoate and 1-hydroxy-2-naphthoate, but it was not transformed by whole cells or their extracts. We conclude that this diol is not responsible for the formation of 1-hydroxy-2-naphthoate from 2-naphthoate but that one of the other three diastereomers is not eliminated as a potential intermediate for a dehydration reaction.

A mutant strain (PL pT 1143) of Pseudomonasputida PL, has been isolated for its inability to growth with p-cymene as carbon source. The mutant oxidizes p-cymene (and p-cumate) to a compound (λmax 293 nm) which is readily converted to 3-hydroxy-p-cumate by acid. 4-Trifluoromethylbenzoate is oxidized by the mutant to an acid-stable intermediate (λmax 277nm) that has been crystallized. The spectral properties (u.v., i.r., NMR and mass) of this metabolite are consistent with those expected for a 2,3-dihydro-2,3-dihydroxy derivative of 4-trifluoromethylbenzoate. Further support of this structure was provided by elemental analysis and the properties of two derivatives of the metabolite, 4-trifluoromethyl-3-hydroxybenzoate and an acetonide formed with 2,2-dimethoxypropane. The stability of a product obtained by treatment of the dihydrodiol metabolite with triacetylosmate indicates that it is the cis-isomer.

Enzymes of the p-cymene pathway in Pseudomonas putida strains cometabolized the intermediate analogue 4-trifluoromethyl(TFM)benzoate. Three products, 4-TFM-2,3-dihydro-2,3-dihydroxybenzoate, 4-TFM-2,3-dihydroxy-benzoate and 2-hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic proterties.Certain TFM-substituted analogue metabolites of the p-cymene pathway were transformed at drastically reduced rates. Hammett type analysis of ring cleavage reactions of 4-substituted 2,3-dihydroxybenzoates revealed the negative inductive and especially mesomeric effect of substituents to be rate determining. Whereas decarboxylation of 3-carboxy-7-TFHOD was not affected by fluorine substitution the subsequent hydrolysis of 7-TFHOD proceeded very slowly. The negative inductive effect of the TFM-group probably inhibited heterolysis of the carbon bond between C5 and C6 of 7-TFHOD.

The oxidation of several mono-hydric phenols by wild type and mutant strains of Pseudomonas aeruginosa T1 has been studied. The data suggest, that a non-specific enzyme sequence of the meta cleavage pathway is induced by all of these phenols, which can catalyze the oxidation of phenol and its analogues to pyruvate, a fatty acid and a carbonyl compound, according to the general scheme of Dagley et al. (1964). Mutants unable to grow on phenol (hydroxylase-negative), have been isolated, and they are also unable to grown on or oxidize the cresols and the xylenols. Revertants of these mutants regain the capacity to grow on all these phenols and are indistinguishable from the wild type. Induced-substrate relationships for the earlier enzymes of the pathway have been determined, e.g., phenol in addition to catechol and the methylcatechols is an inducer for catechol 2,3-oxygenase. Analysis of the enzymic content of cells grown in a variety of steadystate conditions shows (a) that the ratio of the specific activities of the “phenol” hydroxylase and catechol 2,3-oxygenase is constant for each of their analogous substrates; and (b) that induction and catabolite repression of catechol 2,3-oxygenase and the “muconic semialdehyde” hydrolyase are coordinate, but that control of the “phenol” hydroxylase is independent.

Thin sections of the obligate methane oxidizing bacterium, Methanomonas methanooxidans, reveal extensive intracytoplasmic membranes. These always occur in a peripheral arrangement. They appear similar to those in other bacteria which utilize specific energy sources. Their structure and function is discussed.

An inducible 3-hydroxybenzoate 6-hydroxylase has been purified to homogeneity from Pseudomonas aeruginosa . It contains FAD as a prosthetic group. 3-Hydroxybenzoate is quantitatively hydroxylated to give gentisate with equimolar consumptions of NADH and O2. NADPH will substitute as an electron donor, and several aromatic analogues of 3-hydroxybenzoate stimulate reduced nucleotide oxidation by the enzyme with formation of both hydrogen peroxide and hydroxylated products. Of various analogues of 3-hydroxybenzoate, those substituted in 2,4,5 and 6-positions are competent substrates; partial uncoupling of electron flow from hydroxylation with concomitant formation of hydrogen peroxide and “gentisates” occurs. The “natural” product of the reaction, gentisate, is an effector in that it stimulates NADH oxidation with the formation of hydrogen peroxide. 3-hydroxybenzoate 6-hydroxylase thus resembles other flavoprotein hydroxylases in the general regulatory properties dictated by their aromatic substrates, pseudosubstrates or effectors.

2,3-Dihydroxybenzoate is oxidized by extracts of Pseudomonas fluorescens to α-hydroxymuconic semialdehyde and CO2. A noninducing substrate analog, 2,3-dihydroxy-p-toluate, has now been used to determine the site of ring cleavage. 2,3-Dihydroxy-p-toluate is oxidized quantitatively with the consumption of 1 mole of O2, evolution of 1 mole of CO2, and the accumulation of 2,6-diketoheptenoate. The product was characterized by (1) spectral analysis of the isolated acid, (2) ring closure in the presence of NH4+ to form 6-methylpicolinate, and (3) its ready conversion to acetate in 76% yield, by extracts of Ps. aeruginosa T1. It is concluded that enzymic cleavage of the benzenoid nucleus by this oxygenase is between carbon atoms 3 and 4, and the enzyme has been named 2,3-dihydroxybenzoate 3,4-oxygenase.

Spontaneous mutants which acquired the ability to utilize d -allylglycine ( d -2-amino-4-pentenoic acid) and dl - cis -crotylglycine ( dl -2-amino- cis -4-hexenoic acid) but not l -allylglycine or dl - trans -crotylglycine could be readily isolated from Pseudomonas putida mt-2 (PaM1). Derivative strains of PaM1 putatively cured of the TOL (pWWO) plasmid were incapable of forming mutants able to utilize the amino acids for growth; however, this ability could be regained by conjugative transfer of the TOL (pWWO) plasmid from a wild-type strain of mt-2 or of the TOL (pDK1) plasmid from a related strain of P. putida (HS1), into cured recipients. dl -Allylglycine-grown cells of one spontaneous mutant (PaM1000) extensively oxidized dl -allylglycine and dl - cis -crotylglycine, whereas only a limited oxidation was observed toward l -allylglycine and dl - trans -crotylglycine. Cell extracts prepared from PaM1000 cells contained high levels of 2-keto-4-hydroxyvalerate aldolase and 2-keto-4-pentenoic acid hydratase, the latter enzyme showing higher activity toward 2-keto- cis -4-hexenoic acid than toward the trans isomer. Levels of other enzymes of the TOL degradative pathway, including toluate oxidase, catechol-2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-hydroxymuconic semialdehyde dehydrogenase, were also found to be elevated after growth on allylglycine. Whole cells of a putative cured strain, PaM3, accumulated 2-keto-4-pentenoic acid from d -allylglycine, which was shown to be rapidly degraded by cell extracts of PaM1000 grown on dl -allylglycine. These same cell extracts were also capable of catalyzing the dehydrogenation of d - but not l -allylglycine and were further found to metabolize the amino acid completely to pyruvate and acetaldehyde. Differential centrifugation of crude cell extracts localized d -allylglycine dehydrogenase activity to membrane fractions. The results are consistent with a catabolic pathway for d -allylglycine and dl - cis -crotylglycine involving the corresponding keto-enoic acids as intermediates, the further metabolism of which is effected by the action of TOL plasmid-encoded enzymes.

Transposition of chromosomal genes using bacteriophage Mu has been used to obtain a partial order of the nine closely linked genes of the enterochelin-dependent iron transport system of Escherichia coli K-12. Fragments of the ent gene cluster were transposed into the conjugative plasmid RP4 and were characterized by genetic complementation. The partial gene order (entD, fes), entF, fep, entC, ent(ABEG)…lip was derived using six plasmids which carried overlapping parts of the cluster. RP4::ent plasmids carrying different fep alleles were used to investigate the number of fep genes in the cluster, and the fep mutations were shown to belong to a single complementation group. Two restriction fragments, one carrying ent(ABCEG) and the other carrying fep, were cloned in vitro using one of the RP4::ent plasmids as a source of DNA enriched in enterochelin system genes. A further restriction fragment, carrying the three remaining genes, entD, fes and entF was cloned directly from the chromosome. The three restriction fragments collectively cover a region of the chromosome 29 kb in length, indicating that the genes of the enterochelin system are clustered but not contiguous.

Micrococcus strain 12B, grown with phthalate, transformed the phthalate analog, phthalaldehydate (2-formylbenzoate), to 3,4-dihydroxyphthalaldehydate which was isolated and identified as its lactol. An 18 O 2 incorporation experiment indicated that a dioxygenase mechanism was involved. It is proposed by analogy, that phthalate is metabolized through cis -3,4-dihydro-3,4-dihydroxyphthalate and 3,4-dihydroxyphthalate by this bacterium.

Summary 3-Hydroxybenzoate 4-hydroxylase has been purified to homogeneity from extracts from extracts pf Ps . testosteroni . It is a flavoprotein (FAD) which catalyzes the transformation of 3-hydroxybenzoate to protocatechuate with equimolar consumption of NADPH and O 2 . NADH is a poor substitute for NADPH. Several analogues of 3-hydroxybenzoate substituted in the 2,4,5 and 6 positions, act as effectors and substrates for NADPH oxidation but with varying efficiencies of hydroxylation. 2,3-, 2,5-, 3,5-dihydroxybenzoates, 3-hydroxyanthranilate, 2-fluoro-5-hydroxybenzoate and 4-fluoro-3-hydroxybenzoate are competent substrates.

Studies with purified orcinol hydroxylase suggest that, contrary to previous conclusions, the enzymes of the orcinol pathway cannot transform analogous compounds to common metabolites. The substrate analogues of orcinol uncouple electron flow from reduced nicotinamide adenine dinucleotide to oxygen from the hydroxylation reaction catalyzed by orcinol hydroxylase.