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Only 2% of the known natural products with acetylenic bonds are alpha-alkynoates. Their polarized, conjugated triple bond is an optimal target for an enzymic hydration. Therefore they are good substrates for the enzymes involved in metabolism of acetylenic compounds, resulting in products that are suitable for bacterial growth. We isolated a Pseudomonas putida strain growing on 2-butynedioate as well as on propynoate, and determined the metabolic pathways of these two alpha-alkynoates. The triple bonds in both compounds were initially hydrated and 2-ketobutandioate as well as 3-ketopropanoate were formed. These two beta-keto acids were decarboxylated resulting in pyruvate and acetaldehyde, respectively. Pyruvate was further hydrolysed mainly to acetate and formate, whereas minor amounts were reduced to lactate. In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate. Analyses of these metabolic processes were performed by in situ 1H-NMR spectroscopy in 1H2O, although the substrates, propynoate and 2-butynedioate, carried only one or even no detectable protons, respectively. However, while protons from the solvent are incorporated in the course of the pathway, the metabolites can be detected and identified. Therefore a detailed determination of the metabolic process is possible.

The lipase-catalysed acetylation of 2-hydroxymethylpiperidine was carried out in the NMR tube and monitored by 1 H NMR spectroscopy for defined periods of time. Two different acylating agents were used: ethyl acetate in C6D6 and vinyl acetate in C6D6. The lipase employed was porcine pancreatic lipase (PPL). Ethyl acetate in C6D6 was hydrolysed by the enzyme but no formation of acylated product could be detected in these dilute solutions. In case of vinyl acetate as the acyl donor, the reaction did not give the desired N-acylated compound but the corresponding oxazolidine-derivative, which was formed by the reaction of the aminoalcohol and the acetaldehyde. This compound was assigned unambiguously without isolation from the reaction medium by total correlation spectroscopy (TOCSY) and gHSQC experiments.

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.

Biotransformation of benzene and derivatives has been used to accumulate meta-ring fission products of several catechols, including catechol, 3- and 4- methylcatechols, and 3- (trifluoromethyl) catechol. Dilute solutions of these yellow highly absorbing products were used to examine reaction conditions that affect their facile cyclizations with nitrogen nucleophiles, principally NH3. The effects of NH4+/NH3 concentration, pH and temperature on the non-enzymic cyclizations of 2-hydroxymuconic semialdehyde to 2-picolinate and its substituted derivatives have been characterised. As expected, the enzymic product from 3-(trifluoromethyl) catechol cyclised 20 times more slowly than that from 3-methylcatechol under identical reaction conditions. Most of the biological and chemical transformations were studied in very dilute solutions for the convenience of optical absorbance measurements. Because of the known sensitivity of the spectral shifts and extinction coefficients to minor changes in pH between 4 and 8 bo...

The biotransformation of racemic mandelate by a mutant of Pseudomonas putida to give the title compound (1) in high yield and enantiomeric purity is reported. An apparent inconsistency in previous assignments of the absolute configuration of 1 and the corresponding methyl ester is resolved to show that (+)-1 has (5S) stereochemistry.

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.

SUMMARY: Rhodopseudomonas palustris was grown on benzoate, cyclohexanecarboxylate or succinate under anaerobic or aerobic conditions. Studies of oxygen uptake by intact bacteria indicated that cyclohexanecarboxylate was metabolized aerobically by a -oxidation sequence and that cultures grown anaerobically on benzoate also possessed this capacity. Bacteria grown on succinate were able to oxidize octanoate but not alicyclic acids. The enzymes necessary for the -oxidation of cyclohexanecarboxylate appeared to be constitutive in both anaerobic and aerobic bacteria, the only exception being an acyl-CoA synthetase which used benzoate and some alicyclic acids as substrates. This acyl-CoA synthetase differed from the constitutive short-chain fatty acyl-CoA synthetase in that it was induced by anaerobic growth on benzoate or by aerobic growth on cyclohexanecarboxylate.

Fluorochemicals are rare in Nature and fluoroaromatic compounds have not been described as natural products with respect to de novo formation of aryl-fluorine bonds. By contrast many synthetic fluoroaromatic compounds are substrates for several microbial enzymes, particularly oxygenases, and are transformed to previously undescribed fluorochemicals suitable for further modifications by other enzymes systems or by chemical means. These lead to interesting multi-functional molecules, monomers for novel chiral and achiral polymers, chiral intermediates and synthons for some heterocycles and α- amino acids. Wild-type and mutant microbial strains have been used to biotransform some fluoroaromatic compounds in near quantitative yields to novel fluorinated products, as well as other flourophenolics already known by chemical synthesis. Some of the microbial transformations of mono-, di-, tri- and tetrafluoroaromatic compounds are described, and the vast potential of further biotransformations is indicated.

The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.

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.

Biotransformations of 3-fluorophthalic acid have been investigated using blocked mutants of Pseudomonas testosteroni that are defective in the metabolism of phthalic acid (benzene-1,2-dicar-boxyfic acid). Mutant strains were grown with L-glutamic acid in the presence of 3-fluorophthalic acid as inducer of phthalic acid catabolic enzymes. Products that accumulated in the medium were isolated, purified and identified as the fluoroanalogues of those produced from phthalic acid by the same strains. The previously undescribed fluorochemicals cis-3-fluoro-4,5-dihydro-4,5-dihydroxyphthalic acid (VI) and 3-fluoro-4,5-dihydroxyphthalic acid (VII) have been obtained by biotransformation of 3-fluorophthalic acid, and 3-fluoro-5-hydroxyphthalic acid (X) from (VI) by freeze drying. In addition, samples of 2-fluoro-3,4-dihydroxybenzoic acid (2-fluoroprotocatechuic acid, VIII) and 3-fluoro-4,Sdi-hydroxybenzoic acid (5-fluoroprotocatechuic acid, IX) were obtained with a mutant deficient in the ring-fission enzyme, showin...

Bacillus stearothermophilus IC3 degraded the meta cleavage product of catechol, 2-hydroxymuconic semialdehyde, to pyruvate and acetaldehyde via the 4-oxalocrotonate pathway. The pathway was identical to those previously delineated in several mesophilic organisms. However, all the enzymes showed activity at 55 degrees C and other properties (substrate specificities and effects of metal ions) also differed from those displayed by the mesophilic enzymes. All enzymes of this meta cleavage pathway, except the 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase and 4-hydroxy-2-oxovalerate aldolase activities, were induced by growth on phenol.

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.

An apparatus for the continuous and automatic measurement of respiration during growth of micro-organisms in stirred aerated culture is described. The effluent atmosphere from the culture vessels is passed through commercially available oxygen and carbon dioxide gas analyzers, and their electrical output is fed to a multipoint recorder. The apparatus has been used to measure the respiration of Escherichia coli, Pseudomonas aeruginosa, Bacillus thuringiensis, and Methylococcus capsulatus during growth on complex and defined media. In addition, p H values and dissolved oxygen concentrations were measured. Growth of P. thuringiensis in glucose-limited media showed unexpected interruptions in the oxygen consumption curves which resembled diauxic growth; growth of B. thuringiensis in complex media showed similar discontinuities of respiration. These results are explained as the sequential utilization of preferred substrates which, in the case of P. aeruginosa were provided as transient intermediates of glucose.

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.

Publisher Summary Vanillate O-demethylase is an inducible enzyme that catalyzes the O-demethylation of vanillic acid to protocatechuic acid and formaldehyde. This chapter discusses the enzyme assay based on oxygen uptake measurements and spectrophotometric assay. The chapter also discusses identification of protocatechuic acid and formaldehyde as products of demethylase activity. Extracts of bacterial cells induced to oxidize vanillate also contain high levels of protocatechuate 3,4- or 4,5-dioxygenase activity. As O-demethylation is the rate-limiting step, and vanillate O-demethylase is sentitire to traditional enzyme purification techniques, protocatechuate is not readily isolated as the reaction product. However, bacterial extracts that rapidly oxidizes vanillate when supplemented with reduced pyridine nucleotide but that exhibit reduced ring-cleavage activity toward protocatchuate may be obtained using an ultracentrifugation procedure.

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.

SUMMARY: When Micrococcus strain 12B grown on o-phthalate was incubated with 3-methylphthalate, three compounds accumulated. These were shown to be 2-pyrone-3-methyl-4,6-dicarboxylic acid, 3,4-dihydroxy-6-methylphthalic acid, and 5-hydroxy-3-methylphthalic acid, all previously undescribed. A pathway for the formation of these compounds is proposed.

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.

The fine structure of Methylococcus capsulatus is described. Particular emphasis is focused on the intracytoplasmic membrane system which is organized as a stacked array of flattened saccules. Each saccule is limited by a 75 A unit membrane and lies in close apposition to adjacent saccules. Methylococcus capsulatus is an obligate methylotroph whose sole source of carbon and energy is methane (or methanol). In this study methane oxidation is demonstrated for the first time in a cell-free system. Work is in progress to determine the cellular organelles which constitute the particulate fraction responsible for methane oxidation. The possible role of the intracytoplasmic membranes in energy transfer is considered in relation to the functions of stacked membrane arrays in other animal, plant and bacterial systems.

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.

The absolute stereochemistry of the 4-bromo- cis -2,3-dihydroxycyclohexa-4,6-diene-1-carboxylic acid, one of a series of diols produced by P. putida strain JT 107, was shown by X-ray crystal analysis to be (2-R, 3-R).

3,4-, 4,5-and 3,5-Difluoro-3,5-cyclohexadiene- cis -1,2-diol-1-carboxylates were prepared from 3,4-and 3,5-difluorobenzoate by microbial oxidation with a mutant strain of Pseudomonas putida JT 103.