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.     

Primary structure, expression in Escherichia coli, and properties of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from Bacillus megaterium.

A Bacillus megaterium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity was subcloned and sequenced. The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron, J. Bacteriol. 171:4222-4231, 1989). The B. megaterium polypeptide was overexpressed in Escherichia coli, partially purified, and shown to exhibit, like P. denitrificans SUMT, substrate inhibition at uroporphyrinogen III concentrations above 0.5 microM, suggesting a common regulation for aerobic cobalamin-producing organisms.     

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.     

Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata

Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE-Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS-PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS-PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (K(a)=40 microM) was achieved by the cationic detergent, cetyl pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence. They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sites and a conserved C-terminal region common to all known PPOs.     

Purification, characterization, and molecular cloning of tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.     

Purification, characterization, and molecular cloning of acidophilic xylanase from penicillium sp.40

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family xylanases from fungi.     

Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species

An extracellular lipase catalyzing the synthesis of macrocyclic lactones in anhydrous organic solvents was purified to homogeneity from Pseudomonas nov. sp. 109, and characterized. The lipase showed a pI of 5.3 on isoelectric focusing and a Mr of 29,000 +/- 1,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With respect to substrate specificity, optimum chain length for acyl moiety varied depending on the type of reaction catalyzed: C18 in monomer lactone formation, C11 or shorter in dimer lactone formation, and C8 in ester hydrolysis. The amino-terminal 19 amino acid residues of the purified lipase were determined as Ser-Thr-Tyr-Thr-Gln-Thr-Lys-Tyr-Pro-Ile-Val-Leu-Ala-His-Gly-Met-Leu-Gly- Phe, and the gene encoding the lipase was identified by hybridization to a synthetic 20-nucleotide probe, cloned, and sequenced. Nucleotide sequence analysis predicted a 311-amino acid open reading frame, a putative ribosome-binding site, and a 26-amino acid sequence at the amino terminus of the sequence that is not found in the mature protein. This 26-amino acid sequence has many of the characteristics common to known signal peptides. The lipase gene encoded a sequence of Val-Asn-Leu-Ile-Gly-His-Ser-His-Gly-Gly which is very well conserved among lipases, and showed 38-40% overall homology to the amino acid sequences of lipases from Pseudomonas fragie and Pseudomonas cepacia, but showed little homology to those of other lipases, suggesting that some structural features are required for catalyzing macrocyclic lactone synthesis in organic solvents and are restricted to lipases of the Pseudomonas origin.     

Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.     

Purification and characterization of uroporphyrinogen decarboxylase from tobacco leaves

1. Uroporphyrinogen decarboxylase which catalyzes the formationof coproporphyrinogen from uroporphyrinogen is located in thesoluble fraction of tobacco leaves and was purified 72 foldthrough ammonium sulphate precipitation and calcium phosphosphategel absorption. 2. Kinetic studies indicated that the apparentMichaelis constant was 1 ? 10^-6 M for uroporphyrinogen III (pH6.5; 37?C). Uroporphyrinogen III served as a much better substratethan uroporphyrinogen I under the standard conditions of thisstudy. 3. Enzyme activity was inhibited by thiol reagents andheavy divalent cations and was stimulated by some chelatingagents. 4. Both chloride and fluoride salts inhibited the formationof coproporphyrinogen from uroporphyrinogen. ¹Present address: Department of Chemistry, Simon Fraser University,Burnaby 2, British Columbia, Canada. ²Present address: Biology Department, Utah State University,Logan, Utah 84322, U. S. A. (Received June 8, 1974; )     

Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.     

Purification,characterization and molecular cloning of Vibrio fluvialis hemolysin

Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.     

Purification,molecular cloning,and characterization of pyridoxine 4-oxidase from Microbacterium luteolum

Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), which catalyzes the oxidation of PN by oxygen or other hydrogen acceptors to form pyridoxal and hydrogen peroxide or reduced forms of the acceptors, respectively, was purified for the first time to homogeneity from Microbacterium luteolum YK-1 (=Aureobacterium luteolum YK-1). The purified enzyme required FAD for its catalytic activity and stability. The enzyme was a monomeric protein with the subunit molecular mass of 53,000 +/- 1,000 Da. PN was the only substrate as the hydrogen donor. Oxygen, 2,6-dichloroindophenol, and vitamin K3 were good substrates as the hydrogen acceptor. The gene (pno) encoding PN 4-oxidase was cloned. The gene encodes a protein of 507 amino acid residues corresponding to the molecular mass of the subunit. PN 4-oxidase was expressed in Escherichia coli and found to have the same properties as the native enzyme from M. luteolum YK-1. Comparisons of primary and secondary structures with other proteins showed that the enzyme belongs to the GMC oxidoreductase family. M. luteolum YK-1 has four plasmids. The pno gene was found on a chromosomal DNA. Search for genes similar in sequence in other organisms suggested that a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, which harbors two plasmids, has a PN degradation pathway I in chromosomal DNA.     

Purification and properties of uroporphyrinogen III synthase from human erythrocytes

Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria.     

Purification and characterization of glutamine synthetase from the archaebacterium Methanobacterium ivanovi.

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the obligate anaerobic archaebacterium Methanobacterium ivanovi. The 130-fold-purified enzyme was obtained by heat treatment, ion-exchange chromatography, and gel filtration. Like all other eubacterial GSs known so far, the GS of M. ivanovi was found to be a dodecamer of about 600,000 daltons composed of a single type of subunit. The enzyme was stable at 63 degrees C for 10 min and was not sensitive to oxygen. The isoelectric point was 4.6, and the optimum pH of gamma-glutamyltransferase activity was 8.0. The Km values for hydroxylamine, glutamine, and ADP in the transferase reaction were 6.8, 22.7, and 0.35 mM, respectively. L-Methionine-DL-sulfoximine strongly inhibited the activity. Like the GS from gram-positive bacteria, Anabaena sp., several yeasts, and mammals, the enzyme from M. ivanovi was not regulated by adenylylation as demonstrated by snake venom phosphodiesterase treatment. Inhibition of the transferase activity by L-alanine, glycine, L-histidine, and L-tryptophan was observed. L-Glutamine alone or in the presence of AMP did not inhibit the GS synthetic activity. The GS of Methanobacterium ivanovi did not cross-react with a variety of antisera against GS from Escherichia coli, Anabaena strain 7120, or Bacillus megaterium. Archaebacterial GS appears to be structurally and functionally similar to eubacterial GS in gram-positive bacteria.     

Purification, characterization, and cDNA cloning of rice class III chitinase

Several chitinases were expressed in a rice cell suspension culture and detected in the medium. One of them, designated as RCB4, was isolated 248 fold from the culture filtrate to homogeneity by 70% ammonium sulfate precipitation, DEAE-cellulose, CM-cellulose, Sephadex G-75 column chromatography, and native gel slicing. RCB4 had a molecular mass of 32 kDa by SDS-PAGE. The optimum temperature was 40 degrees C, and 96% of its activity still remained at 60 degrees C. The optimum pH was 4, and 95% of its activity was maintained at pH 2. Using a substrate (GlcNAc)6, the Km and Vmax values of RCB4 were 0.53 mM and 11.1 mM/min, respectively. The N-terminal and internal amino acid sequences of RCB4 were determined to be VNSNLFRDYIGA and MALWA, respectively. A cDNA (C12523) clone that contained the N-terminal and internal amino acid sequences of RCB4 was obtained, sequenced, and renamed RCB41. RCB41 encoded 307 amino acid protein with a signal peptide of 25 amino acids and showed a 45% similarity to gladiolus chitinase GBC-a, one of the class III chitinase family. The expression of RCB4l in E. coli showed that RCB41 encodes a chitinase.     

Purification,characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae

Background

In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS).

Methods

The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant.

Results

The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14 kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30 °C and 7.0 respectively. The K_m V_max and K_cat against S-adenosyl-l-methionine (AdoMet) were 4.95 μM, 3.2 U/mg and 6.4 s^− 1 respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a K_i as 10.9 μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan^r exhibited almost 50% reduction in intracellular trehalose concentration.

Conclusion

A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae.

General significance

This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.     

Purification and characterization of an anabolic fumarate reductase from Methanobacterium thermoautotrophicum.

An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the cells of the archaebacterium Methanobacterium thermoautotrophicum. A major portion of the purification was performed inside an anaerobic chamber, employing reducing agents to maintain low redox potentials. The apparent molecular weight of the native enzyme is 78,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a minimal subunit molecular weight of about 20,000. Iodoacetamide (1 mM) and copper chloride (5 mM) caused significant loss in the enzyme activity. The optimum temperature for the enzymatic activity was 75 degrees C. The pH optimum was found to be 7.0. The fumarate reductase had an apparent Km of 0.20 mM for fumarate. Purified enzyme was colorless; spectroscopic studies indicated the absence of flavins as a cofactor. The spectral data, however, suggested the presence of an unknown cofactor tightly bound to the enzyme. Fumarate reductase is involved in the anabolic rather than the catabolic metabolism of M. thermoautotrophicum.