Morpholine-induced formation of L-alanine dehydrogenase activity in Mycobacterium strain HE5.
An NAD-dependent, morpholine-stimulated L-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4. 6-fold increase of L-alanine dehydrogenase activity when L-alanine was supplied at suboptimal concentration. L-alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of L-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent Km values for L-alanine and NAD were 1.0 and 0.2 mM, respectively. Km values of 0. 6, 0.02, and 72 mM for pyruvate, NADH, and NH4+, respectively, were estimated at pH 8.7 for the reductive amination reaction.
Schuffenhauer
G
G
Institut für Mikrobiologie, Martin-Luther-Universität Halle, Kurt-Mothes-Strasse 3, D-06099 Halle, Germany.
Schräder
T
T
Andreesen
J R
JR
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001673:Biodegradation, Environmental; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology*; D009161:Mycobacterium / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment
D001426:Bacterial Proteins; D009025:Morpholines; C037574:morpholine; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase