3-Hydroxybenzoate 6-hydroxylase from Pseudomonas aeruginosa

An inducible 3-hydroxybenzoate 6-hydroxylase has been purified to homogeneity from $\text{Pseudomonas aeruginosa}$. 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.     

Induction of 3-Hydroxybenzoate 2-Hydroxylase in a Pseudomonas testosteroni Mutant

Benzoate was established as the inducer of a unique 3-hydroxybenzoate 2-hydroxylase activity found in a Pseudomonas testosteroni mutant which is unable to grow on m-hydroxybenzoate as its sole source of carbon and energy.     

Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from Pseudomonas putida

4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines. This enzyme was purified to homogeneity from Pseudomonas putida by affinity chromatography. The protein had a molecular weight of 91,000 and was a dimer of identical subunits. It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity. The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2 x 10(-4) M and 5.9 x 10(-5) M respectively. It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.     

Purification and characterization of the 4-hydroxyphenylacetic acid-3-hydroxylase from Pseudomonas putida U

4-Hydroxyphenylacetic acid-3-hydroxylase from Pseudomonas putida U was purified to homogeneity (96-fold) from bacterial cultures grown in a chemically defined medium containing 4-hydroxyphenylacetic acid as the sole carbon source. The maximal rate of catalysis occurred at pH 7.5 and 40°C. Under these conditions, the K_m values calculated for 4-hydroxyphenylacetic acid, NADH and FAD were 38, 41 and 4 μM respectively. The native enzyme (M_r 65 000) had two identical subunits in an α₂ oligomeric structure and required the addition of FAD, so it was classified as an external flavoprotein monooxygenase. 4-Hydroxyphenylacetic acid-3-hydroxylase showed a broad substrate range. It was specifically induced by 4-hydroxyphenylacetic acid, although phenylacetic acid and some phenyl-alkanoic acids also induced enzymatic activity to a lesser extent. 4-Hydroxyphenylacetic acid-3-hydroxylase induction and 4-hydroxyphenylacetic acid consumption were unaffected by the presence of glucose, suggesting that the uptake and hydroxylation of 4-hydroxyphenylacetic acid are not under carbon catabolite repression.     

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.     

Purifications and properties of L-mandelate- 4-hydroxylase from Pseudomonas convexa.

An inducible l-mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of l-mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe²⁺, and O₂ for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with l-mandelate, NADPH, and ferrous sulfate and K_m values for these substrates were found to be 1 × 10^?4, 1.9 × 10^?4, and 4.7 × 10^?5m, respectively. The enzyme is very specific for l-mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.     

Mandelic acid-4-hydroxylase, a new inducible enzyme from Pseudomonas convexa

A soluble fraction of $\text{Pseudomonas convexa}$ catalyzed the hydroxylation of mandelic acid to $\text{p}$-hydroxymandelic acid. The enzyme had a pH optimum of 5.4 and showed an absolute requirement for Fe²⁺, tetrahydropteridine, NADPH. $\text{p}$-Hydroxymandelate, the product of the enzyme reaction was identified by paper chromatography, thin layer chromatography, UV and IR-spectra.     

Cloning of the gene for delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni

We have cloned an approx. 5-kb fragment of Pseudomonas testosteroni DNA containing the structural gene of delta 5-3-ketosteroid isomerase into the EcoRI site of the lambda gt11 genome. Escherichia coli infected with these recombinant phages produce a polypeptide which is recognized by antiserum raised against the purified isomerase. Four of the recombinant lambda gt11 clones contain significant levels of isomerase activity and produce an immunopositive polypeptide of the same apparent Mr as the native isomerase obtained from P. testosteroni. The approx. 5-kb fragment hybridizes to synthetic 21-mer and 17-mer oligodeoxynucleotide mixtures corresponding to the 5' and 3' regions, respectively, of the expected nucleotide sequence of the gene.     

4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: purification of a plant membrane-bound prenyltransferase

Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc.. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K _m values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 μM and 13.8 μM, respectively. Received: 2 July 1997 / Accepted: 14 October 1997     

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.     

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.     

丛毛睾丸酮单胞菌ZD4-1和铜绿假单胞菌ZD4-3降解芳香烃化合物的机理

从某农药厂二沉池污泥中筛选分离得到两株革兰氏阴性的芳香烃降解菌ZD41和ZD43。经鉴定,它们分别属于Comamonas testosteroni和Pseudomonas aeruginosa。基于16S rDNA 序列的系统分类分析,结果表明,在分类地位上菌株ZD41和ZD43 分别属于两个不同的分类亚组。苯酚降解产物紫外光谱扫描和双加氧酶检测证明,菌株ZD41利用邻裂途径降解苯酚,而ZD43则通过间裂途径降解苯酚,邻裂途径的1,2双加氧酶和间裂途径的2,3双加氧酶都是可诱导的双加氧酶,其活性强烈的依赖于降解底物的出现。芳香烃降解试验结果表明,邻裂和间裂两种途径的降解性能不一样,虽然ZD43降解苯酚的效率要高于菌株ZD41,但是ZD41降解苯酚的pH值范围以及芳烃利用基质谱宽于后者。     

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.