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 -