Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli.
The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde:NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322. Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5'-AMP Sepharose, gel-filtration and ion-exchange chromatography. The amino acid composition was determined. The dehydrogenase was found to be a tetramer with identical 55 kDa subunits. Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred. The dehydrogenase was highly specific for betaine aldehyde. None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively. The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K+. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant.
Norwegian College of Fishery Science, University of Tromsø, Norway.
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D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D050728:Betaine-Aldehyde Dehydrogenase; D002412:Cations; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology*; D005590:Fractional Precipitation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009994:Osmolar Concentration; D010957:Plasmids; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014882:Water-Electrolyte Balance
D000596:Amino Acids; D002412:Cations; D013439:Sulfhydryl Reagents; D009243:NAD; D000445:Aldehyde Oxidoreductases; D050728:Betaine-Aldehyde Dehydrogenase