Publications

• Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica.

  Waditee R., Tanaka Y., Aoki K., Hibino T., Jikuya H., Takano J., Takabe T., Takabe T.

  Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacteriu ... >> More

  Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated two N-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. One of gene products (ORF1) catalyzed the methylation reactions of glycine and sarcosine with S-adenosylmethionine acting as the methyl donor. The other one (ORF2) specifically catalyzed the methylation of dimethylglycine to betaine. Both enzymes are active as monomers. Betaine, a final product, did not show the feed back inhibition for the methyltransferases even in the presence of 2 m. A reaction product, S-adenosyl homocysteine, inhibited the methylation reactions with relatively low affinities. The co-expressing of two enzymes in Escherichia coli increased the betaine level and enhanced the growth rates. Immunoblot analysis revealed that the accumulation levels of both enzymes in A. halophytica cells increased with increasing the salinity. These results indicate that A. halophytica cells synthesize betaine from glycine by a three-step methylation. The changes of amino acids Arg-169 to Lys or Glu in ORF1 and Pro-171 to Gln and/or Met-172 to Arg in ORF2 significantly decreased V(max) and increased K(m) for methyl acceptors (glycine, sarcosine, and dimethylglycine) but modestly affected K(m) for S-adenosylmethionine, indicating the importance of these amino acids for the binding of methyl acceptors. Physiological and functional properties of methyltransferases were discussed. << Less

  J. Biol. Chem. 278:4932-4942(2003) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Kinetic mechanism of choline oxidase from Arthrobacter globiformis.

  Gadda G.

  Choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. The enzyme is capable of accepting betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for ... >> More

  Choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. The enzyme is capable of accepting betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to the aldehyde intermediate and of betaine-aldehyde to glycine-betaine. The steady state kinetic mechanism has been determined at pH 7 with choline and betaine-aldehyde as substrate to be sequential, consistent with oxygen reacting with the reduced enzyme before release of betaine-aldehyde or glycine-betaine, respectively. A K(m) value < or =20 microM has been estimated for betaine-aldehyde based on the kinetic pattern with a y-intercept seen in a plot of 1/rate versus 1/[oxygen]. The kinetic data suggest that betaine-aldehyde predominantly remains bound at the active site during turnover of the enzyme with choline. In agreement with such a conclusion, less than 10% betaine-aldehyde has been found in the reaction mixture under enzymatic turnover with saturating concentrations of choline. The k(cat) values were 6.4+/-0.3 and 15.3+/-2.5 s(-1) for choline and betaine-aldehyde, respectively, suggesting that a kinetic step in the oxidation of choline to the aldehyde intermediate must be partially rate-limiting for catalysis. Cleavage of the CH bond of choline as being partially rate-limiting for catalysis is discussed. << Less

  Biochim Biophys Acta 1646:112-118(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning, sequence analysis, and purification of choline oxidase from Arthrobacter globiformis: a bacterial enzyme involved in osmotic stress tolerance.

  Fan F., Ghanem M., Gadda G.

  Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, one of a limited number of compounds that accumulate to high levels in the cytoplasm of cells to prevent dehydration and plasmolysis in adverse hyperosmotic environments. In the present study, the highly GC rich c ... >> More

  Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, one of a limited number of compounds that accumulate to high levels in the cytoplasm of cells to prevent dehydration and plasmolysis in adverse hyperosmotic environments. In the present study, the highly GC rich codA gene encoding for choline oxidase was cloned from genomic DNA of Arthrobacter globiformis strain ATCC 8010 and expressed to high yields in Escherichia coli strain Rosetta(DE3)pLysS. The resulting enzyme was purified to high levels in a single chromatographic step using DEAE-Sepharose, as shown by SDS-PAGE analysis. Denaturation and mass spectroscopic analyses showed that the covalent linkage between the FAD cofactor and the protein is preserved in recombinant choline oxidase, consistent with protein flavinylation being a self-catalytic process. The enzyme was shown to be a homodimer of 120,000 Da by size-exclusion chromatography and to be active with both choline and betaine aldehyde as substrate. Sequencing analysis indicated that the nucleotide sequence of codA originally reported in GenBank contains seven flaws, resulting in a translated protein with a significantly altered amino acid sequence between position 298 and 410. << Less

  Arch. Biochem. Biophys. 421:149-158(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

  Gadda G., Powell N.L., Menon P.

  Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibi ... >> More

  Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase. << Less

  Arch Biochem Biophys 430:264-273(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and characterization of choline oxidase from Arthrobacter globiformis.

  Ikuta S., Imamura S., Misaki H., Horiuti Y.

  Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavo ... >> More

  Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5. << Less

  J Biochem 82:1741-1749(1977) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Choline oxidase, a catabolic enzyme in Arthrobacter pascens, facilitates adaptation to osmotic stress in Escherichia coli.

  Rozwadowski K.L., Khachatourians G.G., Selvaraj G.

  Choline oxidase (EC 1.1.3.17) is a bifunctional enzyme that is capable of catalyzing glycine betaine biosynthesis from choline via betaine aldehyde. A gene (cox) encoding this enzyme in the gram-positive soil bacterium Arthrobacter pascens was isolated and characterized. This gene is contained wit ... >> More

  Choline oxidase (EC 1.1.3.17) is a bifunctional enzyme that is capable of catalyzing glycine betaine biosynthesis from choline via betaine aldehyde. A gene (cox) encoding this enzyme in the gram-positive soil bacterium Arthrobacter pascens was isolated and characterized. This gene is contained within a 1.9-kb fragment that encodes a polypeptide of approximately 66 kDa. Transfer of this gene to an Escherichia coli mutant that is defective in betaine biosynthesis resulted in an osmotolerant phenotype. This phenotype was associated with the ability of the host to synthesize and assemble an enzymatically active choline oxidase that could catalyze biosynthesis of glycine betaine from an exogenous supply of choline. Although glycine betaine functions as an osmolyte in several different organisms, it was not found to have this role in A. pascens. Instead, both choline and glycine betaine were utilized as carbon sources. In A. pascens synthesis and activity of choline oxidase were modulated by carbon sources and were susceptible to catabolite repression. Thus, cox, a gene concerned with carbon utilization in A. pascens, was found to play a role in adaptation to an environmental stress in a heterologous organism. In addition to providing a possible means of manipulating osmotolerance in other organisms, the cox gene offers a model system for the study of choline oxidation, an important metabolic process in both procaryotes and eucaryotes. << Less

  J Bacteriol 173:472-478(1991) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• On the catalytic mechanism of choline oxidase.

  Fan F., Gadda G.

  Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with betaine aldehyde as an intermediate. In this study, primary deuterium and solvent kinetic isotope effects have been used to elucidate the mechanism for substrate oxidation by choline oxidase using both steady ... >> More

  Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with betaine aldehyde as an intermediate. In this study, primary deuterium and solvent kinetic isotope effects have been used to elucidate the mechanism for substrate oxidation by choline oxidase using both steady-state kinetics and rapid kinetics techniques. The D(kcat/Km) value with 1,2-[2H4]-choline at saturating oxygen concentration was independent of pH in the range between 6.5 and 10, with a value of approximately 10.6, indicating that CH bond cleavage is not masked by other titratable kinetic steps belonging to the reductive half-reaction. In agreement with this conclusion, a Dkred value of approximately 8.9 was determined at pH 10 for the anaerobic reduction of the flavin by choline, irrespective of whether aqueous or deuterated solvent was used. At pH 10, both the D2(O)(kcat/Km) and the D2(O)kred values were not different from unity with choline or 1,2-[2H4]-choline, while the Dkcat and D2(O)kcat values were 7.3 and 1.1, respectively. The kcat and kred values were 133 s(-1) and 135 s(-1) with betaine aldehyde and 60 s(-1) and 93 s(-1) with choline. These data are consistent with a chemical mechanism in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage and with chemical steps of flavin reduction by choline and betaine aldehyde being rate limiting for the overall turnover of the enzyme. << Less

  J Am Chem Soc 127:2067-2074(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structural characterization and mapping of the covalently linked FAD cofactor in choline oxidase from Arthrobacter globiformis.

  Rand T., Halkier T., Hansen O.C.

  The flavoenzyme choline oxidase catalyzes the oxidation of choline and betaine aldehyde to betaine. Earlier studies have shown that the choline oxidase from Arthrobacter globiformis contains FAD covalently linked to a histidine residue. To identify the exact type of flavin binding, the FAD-carryin ... >> More

  The flavoenzyme choline oxidase catalyzes the oxidation of choline and betaine aldehyde to betaine. Earlier studies have shown that the choline oxidase from Arthrobacter globiformis contains FAD covalently linked to a histidine residue. To identify the exact type of flavin binding, the FAD-carrying amino acid residue was released by acid hydrolysis. The fluorescence excitation maxima of the isolated aminoacylriboflavin, showing a hypsochromic shift of the near-ultraviolet band relative to riboflavin, and the pH-dependent flavin fluorescence confirmed the presence of an 8alpha-substituted flavin linked to histidine. Similarly, MALDI-TOF mass spectrometry showed a molecular mass corresponding to histidylriboflavin. Classical experiments used to distinguish between the N(1) and N(3) isomers all indicated that the flavin was linked to the N(1) position of the histidine residue. The position of the FAD-carrying histidine residue in the choline oxidase polypeptide was identified by tryptic cleavage of the denatured enzyme, HPLC separation of the proteolytic peptide fragments, and characterization of the purified flavin-carrying peptide by mass spectrometry and spectroscopy. The FAD moiety was assigned to the tryptic peptide, His-Ala-Arg, corresponding to residues 87-89 in the open reading frame of the previously published cDNA sequence. Further analysis of the flavopeptide by collision-induced dissociation mass spectrometry confirmed that the flavin cofactor was attached to His(87). We conclude that this variant of choline oxidase contains 8alpha-[N(1)-histidyl]FAD at position 87 in the polypeptide chain. << Less

  Biochemistry 42:7188-7194(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.