ABSTRACT
An NAD-dependent alcohol-aldehyde oxidoreductase was purified to homogeneity and characterized from cell extracts of the thermophilic microorganism Bacillus acidocaldarius. The 500-fold purified homogeneous enzyme had a molecular mass of 154 kDa, as shown by gel filtration and glycerol gradient centrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis the protein showed one band of 38 kDa, indicating that the enzyme is a tetramer composed of subunits of identical molecular weight. Ethanol was the best substrate with the highest kcat/Km values, and the enzyme showed a substrate specificity that included linear, secondary and cyclic alcohols, as well as anisaldehyde, but it was not active on ketones. The protein contains eight zinc atoms per tetramer, four of which are removed by chelating agents with a concomitant loss of thermal stability. Circular dichroism spectra and determination of the NH2-terminal sequence allowed structural and homology comparison with other alcohol dehydrogenases from animal and bacterial sources.
Subject(s)
Alcohol Dehydrogenase/isolation & purification , Alcohol Dehydrogenase/metabolism , Bacillus/enzymology , Alcohol Dehydrogenase/chemistry , Amino Acid Sequence , Animals , Centrifugation, Density Gradient , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Horses , Hot Temperature , Ketones , Kinetics , Liver , Molecular Sequence Data , Molecular Weight , Sequence Homology, Amino Acid , Substrate Specificity , ThermodynamicsABSTRACT
Reaction of thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus with iodoacetate at pH 9.0 and 37 degrees C significantly increases the oxidation rate of aliphatic and aromatic alcohols and decreases the reduction rate of aromatic aldehydes. The archaeal ADH is chemically modified and activated in a Michaelis-Menten-type reaction, where one molecule of the reagent binds per active site. NAD+ in micromolar concentration protects the enzyme against the inhibitor in an uncompetitive manner, while imidazole significantly increases the extent of the activation. Carboxymethylation selectively modifies one out of five cysteine residues per subunit, namely, Cys 38, located in the catalytic site, as determined by peptide and sequence analysis, and enhances by up to 25-fold the oxidation rate of benzyl alcohol. Carboxymethylated SsADH is less thermostable and shows a temperature optimum 30 degrees C lower than that of the native enzyme. The carboxymethylated enzyme exhibits a lower affinity toward the oxidized and reduced coenzyme. The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 are 60- and 200-fold higher, respectively, compared to the native enzyme. The significant isotope effect in alcohol oxidation suggests that hydride transfer partially limits the turnover rate of the reaction catalyzed by the modified enzyme, whereas the rate-limiting step for the native enzyme is NADH dissociation. Carboxymethylated enzyme probably gives higher maximum velocities of oxidation because of the faster dissociation of the modified enzyme-coenzyme complex.
Subject(s)
Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Sulfolobus/enzymology , Alkylating Agents/pharmacology , Buffers , Cysteine/chemistry , Enzyme Activation/drug effects , Enzyme Stability , Hydrogen-Ion Concentration , Iodoacetates/pharmacology , Iodoacetic Acid , Kinetics , NAD/metabolism , Protein Conformation , TemperatureABSTRACT
The gene coding for the beta-glycosidase from the archaeon Sulfolobus solfataricus has been overexpressed in Escherichia coli. The enzyme was purified to homogeneity with a rapid purification procedure employing a thermal precipitation as a crucial step. The final yield was 64% and the purification from the thermal precipitation was 5.4-fold. The expressed enzyme shows the same molecular mass, thermophilicity, thermal stability, and broad substrate specificity, with noticeable exocellobiase (glucan 1,4-beta-D-glucosidase) activity, of the enzyme purified from S. Solfataricus. We provide evidence that the beta-glycosidase can assume its functional state in E. coli without the contribution of N-epsilon-methylated lysine residues.
Subject(s)
Glycoside Hydrolases/chemistry , Sulfolobus/enzymology , Amino Acid Sequence , Amino Acids/analysis , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Gene Expression Regulation, Bacterial/genetics , Glycoside Hydrolases/genetics , Glycoside Hydrolases/isolation & purification , Glycoside Hydrolases/metabolism , Hydrolysis , Kinetics , Lysine/analogs & derivatives , Lysine/analysis , Molecular Sequence Data , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis , Substrate Specificity , Temperature , ThermodynamicsABSTRACT
The enzyme with beta-galactosidase activity from Sulfolobus solfataricus strain MT-4, like other enzymes of this type isolated from thermophilic sources, has broad specificity for beta-D-gluco-, fuco- and galacto-sides. The beta-galactosidase activity was purified by a new procedure that improved yields (44%) and final specific activity (182 units mg-1 at 75 degrees C using chromogenic beta-D-galactoside as substrate). The enzyme hydrolysed a large number of beta-linked glycoside dimers and oligomers; chromogenic beta-glucosides and beta-fucosides are the preferred substrates, and kinetic analysis indicated that they bind to a common catalytic site. The order of catalytic efficiency was beta 1-3 > beta 1-4 > beta 1-6 and cellotetraose > cellotriose > cellobiose for glucose dimers and oliogomers respectively. The cleavage occurred at the non-reducing end of the oligosaccharide, and the enzyme showed noticeable specificity also for the aglycone part of substrates. From these results the enzyme from S. solfataricus strain MT-4 is defined as a true glycosyl hydrolase with remarkable exo-glucosidase activity and it is designated S beta-gly.
