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.     

beta-agarases I and II from Pseudomonas atlantica. Purifications and some properties

The agarose-degrading system of Pseudomonas atlantica has been re-examined. In addition to the previously reported extracellular endo-beta-agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333-335] a second, membrane-bound endo-enzyme activity, beta-agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose] and also to degrade partially 6-O-methylated agarose to neoagarobiose and 6(1)-O-methyl-neoagarbiose. Novel assays were devised for beta-agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of beta-agarase I and II. beta-Agarase I was purified 670-fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G-100. The enzyme was resolved from the small amount of extracellular beta-agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32000. Activity was observed against agar over the pH range 3.0-9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30 degrees C but only for up to 2 h at 40 degrees C. beta-Agarase II was partially purified (5-fold) from the soluble fraction of disrupted cells by chromatography on Sephadex G-100, hydroxyapatite and DEAE-Sepharose CL-6B. This preparation was free of beta-agarase I and disaccharidase. beta-Agarase II was stimulated by NaCl, optimally in the range 0.10-0.20 mol dm-3 (2.4-fold the activity at 0.010 mol dm-3 NaCl). Alkali earth metal (0.002 mol dm-3 CaCl2 or 0.005 mol dm-3 MgCl2) gave 1.2-fold the normal activity. Optimum activity was over pH 6.5-7.5.     

Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans.

The enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity. 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA. Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.     

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.     

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.     

Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa.

A microorganism capable of degrading DL-mandelic acid was isolated from sewage sediment of enrichment culture and was identified as Pseudomonas convexa. It was found to metabolize mandelic acid by a new pathway involving 4-hydroxymandelic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid as aromatic intermediates. All the enzymes of the pathway were demonstrated in cell-free extracts. L-Mandelate-4-hydroxylase, a soluble enzyme, requires tetrahydropteridine, nicotinamide adenine dinucleotide phosphate, reduced form, and Fe2+ for its activity. The next enzyme, L-4-hydroxymandelate oxidase (decarboxylating), a particulate enzyme, requires flavine adenine dinucleotide and Mn2+ for its activity. A nicotinamide adenine dinucleotide-dependent, as well as a nicotinamide adenine dinucleotide phosphate-dependent, benzaldehyde dehydrogenase has been resolved and partially purified.     

Purification and properties of benzoate-4-hydroxylase from a soil pseudomonad.

Benzoate-4-hydroxylase from a soil pseudomonad was isolated and purified about 50-fold. Polyacrylamide gel electrophoresis of this enzyme preparation showed one major band and one minor band. The approximate molecular weight of the enzyme was found to be 120,000. Benzoate-4-hydroxylase was most active around pH 7.2. The enzyme showed requirements for tetrahydropteridine as the cofactor and molecular oxygen as the electron acceptor. NADPH, NADH, dithiothreitol, β-mercaptoethanol, and ascorbic acid when added alone to the reaction mixture did not support the hydroxylation reaction to any significant extent. However, when these compounds were added together with tetrahydropteridine, they stimulated the hydroxylation. This stimulation is probably due to the reduction of the oxidized pteridine back to the reduced form. This enzyme was activated by Fe²⁺ and benzoate. It was observed that benzoate-4-hydroxylase could catalyze the oxidation of NADPH in the presence of benzoate,p-aminobenzoate, p-nitrobenzoate, p-chlorobenzoate, and p-methylbenzoate, with only benzoate showing maximum hydroxylation. Inhibition studies with substrate analogs and their kinetic analysis revealed that the carboxyl group is involved in binding the substrate to the enzyme at the active center. The enzyme catalyzed the conversion of 1 mol of benzoate to 1 mol of p-hydroxybenzoate with the consumption of slightly more than 1 mol of NADPH and oxygen.     

The Metabolism of Gallic Acid by Pseudomonas convexa X.1

S ummary . Some aspects of gallic acid (3,4,5-trihydroxybenzoic acid) degradation by a bacterial isolate, Pseudomonas convexa X.1, have been investigated. The ability of suspensions of this organism, previously adapted to gallic acid, to oxidize a variety of organic substrates was studied and the results obtained, analyzed with the aid of Stanier's simultaneous adaptation theory in an attempt to identify intermediates of eallic acid metabolism. As a result, α-ketoglutaric acid was suspected of being such an intermediate and was later isolated as its 2,4-dinitrophenylhydrazone during gallic acid metabolism. Gallic acid adapted cells of Ps. convexa X.1 were not able to oxidize the gallate esters and attempts to induce activity were unsuccessful. The usefulness of Stanier's theory for selection of potential metabolites is discussed and a tentative degradative pathway for gallic acid metabolism is proposed.     

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.     

Purification and characterization of 1-naphthol-2-hydroxylase from carbaryl-degrading Pseudomonas strain c4

Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants K(m) and V(max) for 1-naphthol and NADPH were determined to be 9.6 and 34.2 microM and 9.5 and 5.1 micromol min(-1) mg(-1), respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The K(i) for 1-naphthol was determined to be 79.8 microM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.     

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.     

Conversion of L-Hydroxyproline to glutamate by extracts of strains of Pseudomonas convexa and Pseudomonas fluorescens

Summary Three strains of Pseudomonas convexa and three strains of Pseudomonas fluorescens were found able to utilize L-hydroxyproline as sole source of carbon and nitrogen. Sonic extracts of these organisms converted L-hydroxyproline to glutamic acid.     

Purification and properties of L-glutaminase-L-asparaginase from Pseudomonas acidovorans.

An enzyme that catalyzes the hydrolysis of both glutamine and asparagine has been purified to homogeneity from extracts of Pseudomonas acidovorans. The enzyme having a ratio of glutaminase to asparaginase of 1.45:1.0 can be purified by a relatively simple procedure and is stable upon storage. The glutaminase-asparaginase has a relatively high affinity for L-asparagine (Km=1.5 X 10(-5) M) and L-glutamine (Km=2.2 X 10(-5) M) and has a molecular weight of approximately 156,000 the subunit molecular weight being approximately 39,000. Injections of the enzyme produced only slight increases in the survival time of C3H/HE mice carrying the asparagine-requiring 6C2HED Gardner lymphoma and of white Swiss mice carrying the glutamine-requiring Ehrlich lymphoma.     

Purification and properties of pantohenase from Pseudomonas fluorescens.

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.     

A peptidyl dipeptidase-4 from Pseudomonas maltophilia: purification and properties

A peptidyl dipeptidase-4 (bacterial PDP-4) was purified to near homogeneity from a supernatant of Pseudomonas maltophilia extracellular medium. Bacterial PDP-4 is a single-polypeptide-chain enzyme, 82 kDa, with an alkaline isoelectric point. Peptides susceptible to hydrolysis by bacterial PDP-4 include angiotensin 1, bradykinin, enkephalins, atriopeptin 2, and smaller synthetic peptides. N-acylated tripeptides are hydrolyzed, but free tripeptides are not. A free carboxy terminus is required for hydrolysis. Peptides with ultimate and penultimate Pro residues are not hydrolyzed. The enzyme does not require an anion for activity. Bacterial PDP-4 was inhibited by EDTA and the dipeptide Phe-Arg. Thiorphan was an inhibitor only at levels well above those required for inhibition of neutral metalloendopeptidase (NEP), an enzyme for which thiorphan is specific. A second NEP and thermolysin inhibitor, phosphoramidon, did not inhibit bacterial PDP-4. The potent angiotensin-converting enzyme inhibitor lisinopril was not inhibitory. Bacterial PDP-4 is distinguished from a similar enzyme from Escherichia coli, which is not susceptible to EDTA inhibition, and one from Corynebacterium equi, which hydrolyzes free tripeptides. These data indicate that the bacterial PDP-4 catalytic site is unlike those of other enzymes that function either wholly or in part as peptidyl dipeptidases.