Details
| Original language | English |
|---|---|
| Pages (from-to) | 282-289 |
| Number of pages | 8 |
| Journal | Archives of Microbiology |
| Volume | 168 |
| Issue number | 4 |
| Publication status | Published - Sept 1997 |
| Externally published | Yes |
Abstract
Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtills. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plas-mid-encoded gbsA gene under T7∅10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD+ and NADP+ as cofactors, but NAD+ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K, values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD+, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress.
Keywords
- Aldehyde dehydrogenase, Bacillus subtilis, Compatible solutes, Glycine betaine synthesis, Osmoregulation
ASJC Scopus subject areas
- Immunology and Microbiology(all)
- Microbiology
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
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In: Archives of Microbiology, Vol. 168, No. 4, 09.1997, p. 282-289.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine
AU - Boch, Jens
AU - Nau-Wagner, Gabriele
AU - Kneip, Susanne
AU - Bremer, Erhard
N1 - Funding Information: Acknowledgements We appreciate the expert technical assistance of J. Gade and the help of V. Koogle in preparing the manuscript. We thank our colleagues W. Buckel and D. Jahn for their advice throughout this study and are grateful to B. Kempf for critical reading of the manuscript. Financial support was provided by the Deutsche Forschungsgemeinschaft (SFB-395) the Graduier-tenkolleg Enzymchemie, and the Fonds der Chemischen Industrie. Copyright: Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1997/9
Y1 - 1997/9
N2 - Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtills. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plas-mid-encoded gbsA gene under T7∅10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD+ and NADP+ as cofactors, but NAD+ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K, values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD+, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress.
AB - Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtills. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plas-mid-encoded gbsA gene under T7∅10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD+ and NADP+ as cofactors, but NAD+ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K, values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD+, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress.
KW - Aldehyde dehydrogenase
KW - Bacillus subtilis
KW - Compatible solutes
KW - Glycine betaine synthesis
KW - Osmoregulation
UR - http://www.scopus.com/inward/record.url?scp=0030763455&partnerID=8YFLogxK
U2 - 10.1007/s002030050500
DO - 10.1007/s002030050500
M3 - Article
C2 - 9297465
AN - SCOPUS:0030763455
VL - 168
SP - 282
EP - 289
JO - Archives of Microbiology
JF - Archives of Microbiology
SN - 0302-8933
IS - 4
ER -