GLYCINE BETAINE ALDEHYDE DEHYDROGENASE FROM BACILLUS-SUBTILIS - CHARACTERIZATION OF AN ENZYME REQUIRED FOR THE SYNTHESIS OF THE OSMOPROTECTANT GLYCINE BETAINE

Production of the compatible solute glycine betaine from its precursor s choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillu s subtilis, The glycine betaine aldehyde dehydrogenase GbsA is an inte gral part of the osmoregulatory glycine betaine synthesis pathway, We strongly overproduced this enzyme in an Escherichia coli strain that e xpressed a plasmid-encoded gbsA gene under T7 phi 10 control, The reco mbinant GbsA protein was purified 23-fold to apparent homogeneity by f ractionated ammonium sulfate precipitation, ion-exchange chromatograph y on Q-Sepharose, and subsequent hydrophobic interaction chromatograph y on phenyl-Sepharose, Molecular sieving through Superose 12 and sedim entation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was s pecific for glycine betaine aldehyde and could use both NAD(+) and NAD P(+) as cofactors, but NAD(+) was strongly preferred. A kinetic analys is of the GbsA-mediated oxidation of glycine betaine aldehyde to glyci ne betaine revealed K-m values of 125 mu M and 143 mu M for its substr ates glycine betaine aldehyde and NAD(+), respectively. Low concentrat ions 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 synt hesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in v itro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrat ions of this osmolyte. The high salt tolerance and its resistance to i ts own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solu te glycine betaine under conditions of high osmolarity stress.