Degradation of p-toluidine by Pseudomonas testosteroni

Abstract A bacterium, which utilizes p -toluidine as sole source of carbon and energy was isolated from soil. The bacterium was identified as Pseudomonas testosteroni .
From enzymatic studies we propose the following pathway for the degradation of p -toluidine: p -toluidine is oxidatively converted to 4-methyl-catechol, which is then cleaved by a meta -pyrocatechase to 2-hydroxy-5-methyl- cis-cis -muconate semialdehyde.     

Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties.

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.     

Purine degradation in Pseudomonas aeruginosa and Pseudomonas testosteroni.

1. Adenine, hypoxanthine, xanthine and guanine are broken down in Pseudomonas aeruginosa and Pseudomonas testosteroni to allantoin by the concerted action of the enzymes adenine deaminase, guanine deaminase, NAD+-dependent xanthine dehydrogenase and uricase. 2. Uric acid is broken down by an unstable, membrane-bound uricase with an unusually low pH optimum. 3. In both strains adenine inhibits growth and xanthine dehydrogenase. A second type of inhibition is manifest only in Ps. testosteroni and concerns the regulation of the biosynthesis of amino acids of the aspartate family. Enzymic studies showed that in this strain aspartate kinase is inhibited by AMP.     

The metabolism of protocatechuate by Pseudomonas testosteroni

1. Protocatechuate 4,5-oxygenase, purified 21-fold from extracts of Pseudomonas testosteroni, was examined in the ultracentrifuge and assigned a mol.wt. of about 140000. 2. When diluted, the enzyme rapidly lost activity during catalysis. Inactivation was partially prevented by l-cysteine. 3. With a saturating concentration of protocatechuate (1·36mm), K_m for oxygen was 0·303mm. This value is greater than the concentration of oxygen in water saturated with air at 20°. 4. Cell extracts converted protocatechuate into γ-carboxy-γ-hydroxy-α-oxovalerate, which was isolated as its lactone. 5. γ-Carboxy-γ-hydroxy-α-oxovalerate pyruvate-lyase activity was stimulated by Mg²⁺ ions and mercaptoethanol. Cells grown with p-hydroxybenzoate as carbon source contained higher concentrations of this enzyme than those grown with succinate.     

Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Location and regulation of expression.

Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.     

3-Hydroxybenzoate 4-hydroxylase from Pseudomonas testosteroni

3-Hydroxybenzoate 4-hydroxylase has been purified to homogeneity from extracts pf $\text{Ps. testosteroni}$. It is a flavoprotein (FAD) which catalyzes the transformation of 3 -hydroxybenzoate to protocatechuate with equimolar consumption of NADPH and O₂. 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.     

Pseudomonas alcaligenes andPseudomonas testosteroni: Characterization and identification

Procedures used in most clinical laboratories do not clearly distinguish betweenPseudomonas alcaligenes andPseudomonas testosteroni. In an examination of 75 features of 31 strains, we found that only microscopic morphology definitively distinguished these two species.Pseudomonas alcalgenes is phenotypically heterogeneous;P. testosteroni is relatively homogeneous. Several additional features will distinguish most strains ofP. alcaligenes.     

Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.     

Testosterone-dependent oxygen consumption in membrane vesicles of Pseudomonas testosteroni

Oxygen consumption was measured in membrane vesicles of Pseudomonas testosteroni using conditions similar to those identified for testosterone transport in these vesicles. Testosterone and NAD⁺, which are primary requirements for testosterone transport, were both required for maximum oxygen consumption suggesting that testosterone transport and oxygen consumption were linked. Testosterone-dependent oxygen consumption was inhibited by 95% by 1 mM KCN indicating that the electron-transport chain could be involved in this process. Respiration appears to play an important role in the transport of steroids by membrane vesicles of P. testosteroni.     

Isolation and study of a bacteriophage of Pseudomonas testosteroni

A virulent phage specific for Pseudomonas testosteroni is described. This phage have a regular icosahedral head (52 nm between opposite angles) and a contractile tail (165 X 8 nm) but no fibers on. The buoyant density is 1,51 +/- 0,01 g/ml. The nucleic acid is an desoxyribonucleic acid with a density of 1,696 +/- 0,03 g/ml and a GC% between 33,7 and 39,7.     

Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases

Pseudomonas putida produces two lipoamide dehydrogenases, LPD-glc and LPD-val. LPD-val is specifically required as the lipoamide dehydrogenase of branched-chain keto acid dehydrogenase and LPD-glc fulfills all other requirements for lipoamide dehydrogenase. Both proteins are dimers with one FAD per subunit. LPD-glc has an absorption maximum at 455 nm, but LPD-val has a maximum at 460 nm. Comparison of amino acid compositions revealed that LPD-glc was more closely related to Escherichia coli and pig heart lipoamide dehydrogenase than to LPD-val. LPD-val did not appear to be closely related to any of the proteins compared with the possible exception of mercuric reductase.     

Membrane-bound respiratory chain of Pseudomonas aeruginosa grown aerobically.

The electron transport chain of the gram-negative bacterium Pseudomonas aeruginosa, grown aerobically, contained a number of primary dehydrogenases and respiratory components (soluble flavin, bound flavin, coenzyme Q9, heme b, heme c, and cytochrome o) in membrane particles of the organism. Cytochrome o, about 50% of the b-type cytochrome, seemed to function as a terminal oxidase in the respiratory chain. The electron transport chain of P. aeruginosa grown aerobically was suggested to be lined up in order of primary dehydrogenase, b, c1, c, o, and oxygen.     

Enzymatic Transformation of Morphine by Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni

Eznyme preparations from Pseudomonas testosteroni containing alpha- and beta- hydroxysteroid dehydrogenases catalyzed the oxidation of morphine and codeine by nicotinamide adenine dinucleotide. Morphine was converted in relatively low yield into 14-hydroxymorphinone probably via morphinone as an intermediate. Codeine was converted to codeinone and 14-hydroxycodeinone. Only the conversions at the 6-position were carred out by the hydroxysteroid dehydrogenase. Hydroxylation at the 14-position did occur spontaneously (or enzymatically with a contaminating enzyme) ater oxidation at the 6-position.     

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.     

Purification and properties of malate dehydrogenase from Pseudomonas testosteroni.

Nicotinamide adenine dinucleotide-linked malate dehydrogenase has been purified from Pseudomonas testosteroni (ATCC 11996). The purification represents over 450-fold increase in specific activity. The amino acid composition of the enzyme was determined and found to be quite different from the composition of the malate dehydrogenases from animal sources as well as from Escherichia coli. Despite this difference, however, the data show that the enzymatic properties of the purified enzyme are remarkably similar to those of other malate dehydrogenases that have been previously studied. The Pseudomonas enzyme has a molecular weight of 74,000 and consists of two subunits of identical size. In addition to L-malate, the enzyme slowly oxidizes other four-carbon dicarboylates having an alpha-hydroxyl group of S configuration such as meso- and (-) tartrate. Rate-determining steps, which differ from that of the reaction involving L-malate, are discussed for the reaction involving these alternative substrates. Oxidation of hydroxymalonate, a process previously undetected with other malate dehydrogenases, is demonstrated fluorometrically. Hydroxymalonate and D-malate strongly enhance the fluorescence of the reduced nicotinamide adenine dinucleotide bound to the enzyme. The enzyme is A-stereospecific with respect to the coenzyme. Malate dehydrogenase is present in a single form in the Pseudomonas. The susceptibility of the enzyme to activation or inhibition by its substrates-particularly the favoring of the oxidation of malate at elevated concentrations-strongly resembles the properties of the mitochondrial enzymes. The present study reveals that whereas profound variations in chemical composition have occurred between the prokaryotic and eukaryotic enzymes, the physical and catalytic properties of malate dehydrogenase, unlike lactate dehydrogenase, are well conserved during the evolutionary process.