Purification and properties of an amine dehydrogenase from Pseudomonas AM1 and its role in growth on methylamine

1. Whole cells of Pseudomonas AM1 grown on methylamine oxidize methylamine, formaldehyde and formate. Crude extracts oxidize methylamine only if supplemented with phenazine methosulphate. 2. By using a spectrophotometric assay, the methylamine-oxidizing enzyme has been purified 20-fold in 31% yield. 3. The enzyme is a dehydrogenase, unable to utilize oxygen, NAD, NADP, flavines or menadione as electron acceptors, but able to utilize phenazine methosulphate, ferricyanide, cytochrome c or brilliant cresyl blue. 4. The enzyme is non-specific, readily oxidizing aliphatic monoamines and diamines, histamine and ethanol-amine. Secondary and tertiary amines, quaternary ammonium salts and aromatic amines are not oxidized. 5. The pH optima for methylamine, n-pentylamine and putrescine are respectively 7.6, 8.0 and 8.5. 6. The K(m) value for methylamine is 5.2mum and that for phenazine methosulphate 56mum. 7. The enzyme will withstand heating for 15min. at 80 degrees without loss of activity, but is inactivated at higher temperatures. It is not inactivated by any pH value between 2.6 and 10.6. 8. The dehydrogenase is inhibited by semicarbazide (K(i) 3.35mum), isoniazid (K(i) 1.17mum), cuprizone (K(i) 0.49mum), p-chloromercuribenzoate (K(i) 0.45mm) and quinacrine (K(i) 12.1mm). 9. The enzyme is absent from succinate-grown cells, and, during adaptation from succinate to methylamine, activity appears before growth on methylamine begins.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Purification and properties of N-acetylneuraminate lyase from Escherichia coli

N-Acetylneuraminate lyase [N-acetylneuraminic acid aldolase EC 4.1.3.3] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase. The enzyme catalyzed the cleavage of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of N-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60 degrees C. The thermal stability increased up to 75 degrees C in the presence of pyruvate. No metal ion was required for the enzyme activity, but heavy metal ions such as Ag+ and Hg2+ were potent inhibitors. Oxidizing agents such as N-bromosuccinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p-chloromercuribenzoic acid and mercuric chloride were also potent inhibitors. The Km values for N-acetylneuraminic acid and N-glycolylneuraminic acid were 3.6 mM and 4.3 mM, respectively. Pyruvate inhibited the cleavage reaction competitively; Ki was calculated to be 1.0 mM. In the condensation reaction, N-acetylglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace N-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, beta-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate. The molecular weight of the native enzyme was estimated to be 98,000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6 M guanidine-HCl, the molecular weight was reduced to 33,000, indicating the existence of 3 identical subunits. The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.     

Purification and properties of 3-ketovalidoxylamine A C-N lyase from Flavobacterium saccharophilum

3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36,000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(beta-aminoethylether)-N,N'-tetraacetic acid and the inhibition was reversed by Ca2+ ion. The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-alpha-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-beta-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24 mM and 0.5 mM, respectively.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph

A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.     

Purification and properties of isocitrate lyase from flax seedlings.

Isocitrate lyase has been purified from flax (Linum usitatissimum) seedlings. The final preparation was homogeneous by the criteria of polyacrylamide disc gel electrophoresis, immunodiffusion, and immunoelectrophoresis. From exclusion chromatography on Sephadex G-200, the molecular weight and Stoke's radius of the enzyme were 264,000 and 5.28 × 10^?7 cm, respectively. The subunit molecular weight was 67,000. Thus, the enzyme appears to be tetrameric. The enzyme required Mg²⁺ and cysteine for activity. The optimal pH of the enzyme was 7.5 both in Tris and in phosphate buffers. There are three disulfide bridges and two of eight cysteine residues are buried. Inactivation of isocitrate lyase resulted from short-term modification of enzymatic thiols but this could be reversed by added thiols. The enzyme was competitively inhibited by glyoxylate, l-tartrate, and malonate in catalysis of isocitrate cleavage.     

Purification and properties of citrate lyase from Streptococcus diacetilactis.

Citrate lyase from Streptococcus diacetilactis has been purified to yield a protein that was homogeneous as judged by sedimentation velocity and sedimentation equilibrium experiments. The enzyme's sedimentation coefficient is 16.8 S and its molecular weight is around 585,000. It contains three nonidentical subunits of about 53,000, 34,000, and 10,000 daltons. The enzyme in its active form contains an acetyl group which turns over during the citrate cleavage reaction. Removal of the acetyl group inactivates the enzyme. The deacetyl enzyme can be partially reactivated by acetylation with acetic anhydride. The enzyme undergoes slow "reaction-inactivation." The rate of inactivation is first order and the rate constant of inactivation is much lower than that for a similar inactivation process of the citrate lyase from Klebsiella aerogenes. Like the latter enzyme it contains stoichiometric amounts of phosphopantothenate. The enzyme is inactivated at pH greater than 8.1 and the presence of citrate provides protection against this inactivation. Sedimentation studies of the enzyme at pH 8.7 indicate that the enzyme is dissociated, which may account for the inactivation. The enzyme is immunologically different from citrate lyases of K. aerogenes and Escherichia coli.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 °C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected ³²P_i was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.     

Purification and properties of bromoperoxidase from Pseudomonas pyrrocinia

A bromoperoxidase was purified and partially characterized from Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. The purified enzyme preparation was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular mass of the enzyme was estimated to be 154 kDa +/- 3 kDa as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band with the mobility of a 76-kDa species. Therefore, in solution at neutral pH, bromoperoxidase exists as a dimeric species. The isoelectric point was 5.0. The prosthetic group of this procaryotic bromoperoxidase was ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. The purified enzyme showed brominating as well as peroxidase and catalase activity.