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 dehydrogena...

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Bibliographic Details
Published in:Archives of microbiology 1999-05, Vol.171 (6), p.417-423
Main Authors: SCHUFFENHAUER, G, SCHRÄDER, T, ANDREESEN, J. R
Format: Article
Language:English
Subjects:
Alanine Dehydrogenase
Amino Acid Oxidoreductases - genetics
Amino Acid Oxidoreductases - isolation & purification
Amino Acid Oxidoreductases - metabolism
Amino Acid Sequence
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Bacterial Proteins - metabolism
Bacteriology
Biodegradation, Environmental
Biological and medical sciences
Electrophoresis, Polyacrylamide Gel
Fundamental and applied biological sciences. Psychology
Genes, Bacterial
Metabolism. Enzymes
Microbiology
Molecular Sequence Data
morpholine
Morpholines - pharmacology
Mycobacterium
Mycobacterium - drug effects
Mycobacterium - enzymology
Mycobacterium - genetics
pyruvic acid
Sequence Alignment
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Summary: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.
ISSN:0302-8933
1432-072X
DOI:10.1007/s002030050728