The doctoral dissertations of the former Helsinki University of Technology (TKK) and Aalto University Schools of Technology (CHEM, ELEC, ENG, SCI) published in electronic format are available in the electronic publications archive of Aalto University - Aaltodoc.
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Pathways of Glycine Betaine Synthesis in Two Extremely Halophilic Bacteria, Actinopolyspora Halophila and Ectothiorhodospira Halochloris

Antti Nyyssölä

Dissertation for the degree of Doctor of Science in Technology to be presented with due permission of the Department of Chemical Technology for public examination and debate in Auditorium KE 2 (Komppa Auditorium) at Helsinki University of Technology (Espoo, Finland) on the 26th of October, 2001, at 12 noon.

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Abstract

Glycine betaine is a solute which is able to restore and maintain the osmotic balance of living cells. In this thesis, the glycine betaine synthesis in two extremely halophilic bacteria Actinopolyspora halophila and Ectothiorhodospira halochloris is investigated.

A. halophila synthesized remarkably high intracellular concentrations of glycine betaine. The highest glycine betaine concentration, determined at 24% (w/v) NaCl, was 33% of the cellular dry weight.

The data presented in this work indicate that the de novo synthesis of glycine betaine proceeds via the threefold methylation of glycine. S-adenosylmethionine acts as the methyl group donor in the reactions. The genes encoding this pathway were cloned and successfully expressed in Escherichia coli. In E. halochloris, glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) catalyze the reaction sequence. In A. halophila all three methylation reactions appear to be catalyzed by a fusion protein. The methyltransferases from the two bacteria show high sequence homology.

Furthermore, it was demonstrated that in addition to the glycine methylation pathway, A. halophila has the ability to oxidize choline to glycine betaine. Choline was first oxidized to betaine aldehyde in a reaction in which H2O2-generation and oxygen consumption are coupled. Betaine aldehyde was oxidized further to glycine betaine in a reaction in which NAD(P)+ was reduced.

The GSMT and SDMT of E. halochloris were expressed in E. coli, purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificity and pH optima near physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent Km and Vmax values were determined for all substrates, when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Glycine betaine inhibited the methylation reactions only at high concentrations.

Finally, it was demonstrated that the expression of the E. halochloris methyltransferase genes in E. coli results in glycine betaine accumulation and improves salt tolerance.

This thesis consists of an overview and of the following 4 publications:

  1. Nyyssölä, A. and Leisola, M., Actinopolyspora halophila has two separate pathways for betaine synthesis, Arch. Microbiol. 2001 (in press).
  2. Nyyssölä, A., Kerovuo, J., Kaukinen, P., von Weymarn, N. and Reinikainen, T., Extreme halophiles synthesize betaine from glycine by methylation, J. Biol. Chem. 275 (2000) 22196-22201.
  3. Nyyssölä, A., Reinkainen, T. and Leisola, M., Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, Appl. Environ. Microbiol. 67 (2001) 2044-2050.
  4. von Weymarn, N., Nyyssölä, A., Reinikainen, T., Leisola, M. and Ojamo, H., Improved osmotolerance of recombinant Escherichia coli by de novo glycine betaine biosynthesis, Appl. Microbiol. Biotechnol. 55 (2001) 214-218.

Keywords: Actinopolyspora halophila, choline, choline oxidation, compatible solutes, Ectothiorhodospira halochloris, dimethylglycine, extreme halophiles, glycine betaine, glycine methylation, halophilism, methyltransferases, sarcosine

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© 2002 Helsinki University of Technology

Last update 2011-05-26