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An NAD+-dependent alanine dehydrogenase from a methylotrophic bacterium.
The Biochemical Journal
|June 15, 1987
Summary
Pseudomonas sp. strain MA produces alanine dehydrogenase for ammonia assimilation and alanine breakdown. This enzyme is highly specific for ammonia and NAD+, with a pH optimum of 9.0.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- Methylotrophic bacteria like Pseudomonas sp. strain MA utilize diverse carbon and nitrogen sources.
- Alanine dehydrogenase (EC 1.4.1.1) plays a role in amino acid metabolism.
Purpose of the Study:
- To characterize the NAD+-dependent alanine dehydrogenase from Pseudomonas sp. strain MA.
- To elucidate the enzyme's specificity, structure, and regulatory properties.
Main Methods:
- Enzyme purification (400-fold).
- Enzyme activity assays with various substrates and cofactors.
- Determination of molecular weight (native and SDS-PAGE).
- pH optimum determination.
- Enzyme induction and repression studies.
Main Results:
- The purified enzyme showed high specificity for ammonia (NH3) and NAD+.
- Hydroxypyruvate and bromopyruvate partially replaced pyruvate.
- Native enzyme Mr was 217,000; SDS-PAGE indicated a four-subunit structure (Mr 53,000 each).
- The enzyme exhibited a pH optimum of 9.0 and functioned primarily in the aminating direction.
- Enzyme expression was induced by ammonia or alanine and repressed by methylamine or glutamate.
Conclusions:
- The alanine dehydrogenase from Pseudomonas sp. strain MA is a tetrameric enzyme with specific substrate requirements.
- The enzyme's activity and regulation suggest dual roles in ammonia assimilation and alanine catabolism within the bacterium.