Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.

S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.     

Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the cobL gene product.

A protein catalyzing methylation at C-5 and C-15 and decarboxylation of the acetic acid side chain at C-12 on precorrin-6y to yield precorrin-8x was purified to homogeneity from a recombinant strain of Pseudomonas denitrificans. It was sequenced at the N terminus and shown to be encoded by the cobL gene.     

Genetic and sequence analysis of an 8.7-kilobase Pseudomonas denitrificans fragment carrying eight genes involved in transformation of precorrin-2 to cobyrinic acid.

A 8.7-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and the genetic analysis revealed that this fragment carries eight different cob genes (cobF to cobM). Six of these genes have the characteristics of translationally coupled genes. cobI has been identified as S-adenosyl-L-methionine (SAM):precorrin-2 methyltransferase structural gene because the encoded protein has the same NH2 terminus and molecular weight as those of the purified enzyme. From protein homology with CobA and CobI, two SAM-dependent methyltransferases of the cobalamin pathway, it is proposed that cobF, cobJ, cobL, and cobM code for other methyltransferases involved in the cobalamin pathway. In addition, purified CobF protein has affinity for SAM, as expected for a SAM-dependent methyltransferase. Accumulation of cobalamin precursors in Agrobacterium tumefaciens mutants complemented by any of these eight genes suggest that, apart from cobI, whose function is identified, the products of all these genes are implicated in the conversion of precorrin-3 into cobyrinic acid.     

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene.

Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-3H]S-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr congruent to 27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr congruent to 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 +/- 0.2 microM for precorrin-6x and 23.5 +/- 3.5 microM for NADPH and a Vmax value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand.     

Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase DNA fragment containing five cob genes and identification of structural genes encoding S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase and cobyrinic acid a,c-diamide synthase.

A 5.4-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment carries five different cob genes (cobA to cobE). Four of these genes present the characteristics of translationally coupled genes. cobA has been identified as the structural gene of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) because the encoded protein has the same NH2 terminus and molecular weight as those determined for the purified SUMT. For the same reasons the cobB gene was shown to be the structural gene for cobyrinic acid a,c-diamide synthase. Genetic and biochemical data concerning cobC and cobD mutants suggest that the products of these genes are involved in the conversion of cobyric acid to cobinamide.     

Isolation and properties of cytochrome c peroxidase from Pseudomonas denitrificans.

The isolation of cytochrome c peroxidase, cytochrome c4, cytochrome c-551 and azurin from Pseudomonas dentrificans is described. The peroxidase has a molecular weight of 63,000 and an isoelectric point of 5.6. Its absorption spectrum suggests that it contains two haem c groups/molecule. Preliminary steady-state kinetic data are reported with cytochromes c-551 and c4 and azurin as the second substrate.     

Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii.

An S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity has been identified in Methanobacterium ivanovii and was purified 4,500-fold to homogeneity with a 38% yield. The enzyme had an apparent molecular weight of 58,200 by gel filtration and consisted of two identical subunits of Mr 29,000, as estimated by gel electrophoresis under denaturing conditions. The Km value for uroporphyrinogen III was 52 nM. The enzyme catalyzed the two C-2 and C-7 methylation reactions converting uroporphyrinogen III into precorrin-2. Unlike Pseudomonas denitrificans SUMT, the only SUMT characterized to date (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet and B. Cameron, J. Bacteriol. 171:4222-4231, 1989), M. ivanovii SUMT did not show substrate inhibition at uroporphyrinogen III concentrations of up to 20 microM. Oligonucleotide probes from limited peptide sequence information were used to clone the corresponding gene. The encoded polypeptide showed more than 40% strict homology with P. denitrificans SUMT. The M. ivanovii SUMT structural gene is likely to be, as is P. denitrificans cobA, involved in corrinoid synthesis.     

Partial purification and characterization of an N2-guanine RNA methyltransferase from chicken embryos.

An N2-guanine RNA methyltransferase has been purified 1000-fold from chick embryo homogenates by phosphocellulose chromatography followed by chromatography on S-adenosylhomocystein-Sepharose. The enzyme was shown to methylate the G10 position of Escherichia coli B tRNAPhe and has a Km of 3X10(-7) M for tRNAPhe and 1.38 X 10(-6) M for S-adenosylmethionine. The molecular weight was estimated to be 77 000 by gel filtration and the pH optimum was 8.0 to 8.5. Magnesium ion was not required for activity but it stimulated the rate of methylation 1.5-fold with an optimum at 12 mM. Ammonium ion stimulated activity about twofold with an optimum at about 83 mM. Sodium and potassium ions above 0.1 M were inhibitory.     

Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.     

Sterol methyltransferase2: purification, properties, and inhibition

Expression of the Arabidopsis sterol methyltransferase2 (SMT2) cDNA in Escherichia coli yields a native protein, when purified to homogeneity, has the predicted molecular mass ca. 40 kDa on SDS-PAGE and recognizes native sterols synthesized by Arabidopsis with a Delta(24(25))-bond (cycloartenol; K(m) 35 microM and k(cat) 0.001s(-1)) and Delta(24(28))-bond (24(28)-methylenelophenol; K(m) 28 microM and k(cat) 0.01 s(-1)). Cycloartenol was converted to a single olefinic product-24(28)-methylenecycloartanol whereas 24(28)-methylenelophenol was converted to a mixture of three stereochemically related products with the Delta(24(28))Z-ethylidene, Delta(24(28))E-ethylidene, and Delta(25(27))-24 beta-ethyl side chains. Structural determinants essential to activity were the nucleophilic features at C-3 and C-24. The double bond position in the sterol substrate influenced catalytic efficiency according to the order: side chain, Delta(24(24))相似文献   

The purification and properties of L-histidine--2-oxoglutarate aminotransferase from Pseudomonas testosteroni.

1. Inducible L-histidine--2-oxoglutarate aminotransferase was purified some 170-fold from extracts of Pseudomonas testosteroni. 2. The preparation showed only one major component after electrophoresis on polyacrylamide gels, though additional minor bands were observed when samples concentrated on a DEAE-cellulose column were used. 3. The molecular weight of the enzyme was found to be approx. 70000 by chromatography on Sephadex G-200. 4. The purification scheme produced enzyme that was inactive in the absence of pyridoxal 5'-phosphate. 5. The equilibrium constant for the reaction L-histidine+2-oxoglutarate equilibrium imidazolylpyruvate+L-glutamate was 0.49. 6. The reaction mechanism was Ping Pong. 7. The enzyme was shown to have only low activity towards aromatic amino acids and was highly specific for 2-oxoglutarate.     

Assay for S-adenosylmethionine: methionine methyltransferase

A quantitative assay for S-adenosylmethionine: methionine methyltransferase in phosphate buffer extracts has been developed. This enzyme catalyzes the biosynthesis of S-methylmethionine from methionine and S-adenosylmethionine. The radioactively labeled product, S-methylmethionine, is first separated from the radioactively labeled substrate, l-methionine, by means of ion-exchange chromatography. Once separated thusly, the amount present can then be directly determined by the use of a liquid scintillation spectrometer.     

Overproduction and purification of the M.HhaII methyltransferase from Haemophilus haemolyticus

The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside results in overproduction of the methyltransferase to about 3% of total cellular protein. The methyltransferase was purified to near electrophoretic homogeneity by phosphocellulose, DEAE, and gel chromatography. Its monomer Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25 kDa, in good agreement with that predicted from the nucleotide sequence. Crystals of the methyltransferase were obtained in the presence of a two-fold molar excess of the duplex oligodeoxynucleotide substrate 5'd-GGACTCC.CCTGAGG.     

Purification and characterization of a monohalomethane-producing enzyme S-adenosyl-L-methionine: halide ion methyltransferase from a marine microalga, Pavlova pinguis.

A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The Km for iodide and SAM were 12 mM and 12 microM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I-, Br-, and Cl- to corresponding monohalomethanes and of bisulfide to methyl mercaptan.     

Comparison of dentrification by Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa.

The course of denitrification of nitrate, nitrite and both compounds together by static cultures of Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa was studied. These strains represent three different types of denitrification: 1. reduction of nitrate to gaseous nitrogen without accumulation of nitrite (P. denitrificans); 2. partial accumulation of nitrite in growing cultures during reduction of nitrate to gaseous nitrogen (P. aeruginosa) and 3. two-phase denitrification that includes reduction of nitrates at the very beginning of the process, and then, after depletion of the former, the reduction of nitrates to gaseous nitrogen (P. stutzeri). These observations differ from the results reported in the literature and possible reasons are discussed.