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Dissertation
Bacterial sarcosine oxidase: comparison of two multisubunit enzymes containing both covalent and noncovalent flavin
Doctor of Philosophy (Ph.D.), Hahnemann University
Jun 1987
DOI:
https://doi.org/10.17918/00009616
Abstract
Sarcosine oxidase from Corynebacterium sp. U-96, a soil organism isolated in Japan, is the first example of a flavoprotein containing both covalently bound flavin and noncovalently bound flavin (Hayashi et al., 1980, 1982; Suzuki, 1981). To determine whether the enzyme from the Japanese Corynebacterium was unique, the isolation of another corynebacterium which produces sarcosine oxidase was attempted. A Corynebacterium (Corynebacterium sp. P-1), isolated by a serial enrichment technique from Philadelphia soil samples, synthesizes sarcosine oxidase in large amounts when grown with sarcosine as sole carbon source. The enzyme, purified to electrophoretic homogeneity, contains 1 mol of noncovalently-bound flavin (flavin adenine dinucleotide (FAD)) plus 1 mol of covalently-bound flavin (8[alpha]-(N3-histidyl)-FAD) per mole of enzyme (Mr, 168,000). The flavin composition of the Philadelphia enzyme is identical to that of the Japanese enzyme. The two flavins appear to have different roles in catalysis. The Philadelphia enzyme, purified in the presence of protease inhibitors, exhibits an unusual quaternary structure, containing four dissimilar subunits (Mr, 100,000, 42,000, 20,000, and 6,000). The covalent flavin is attached to the next to the largest subunit. The same subunit composition is observed in Western blot analysis, when cell extracts of Corynebacterium sp. P-1 prepared in the presence of trichloroacetic acid are probed by a sarcosine oxidase specific antibody, indicating that the subunits are a genuine property of the enzyme as it exists in vivo. Enzymes containing 4 or more nonidentical subunits frequently exhibit complex functions, suggesting that sarcosine oxidase might have a broader metabolic role in corynebacteria. The fate of formaldehyde, produced during the oxidation of sarcosine, was investigated and shown to have a role in one-carbon metabolism. The enzyme binds tetrahydrofolate and, in the presence of sarcosine, catalyzes the synthesis of 5,10-methylenetetrahydrofolate. The enzymes exhibit many similarities but are distinguishable in electrophoretic studies. Immunologically, the enzymes were shown to be similar but not identical. The results indicate that the synthesis of a sarcosine oxidase containing both covalent and noncovalent flavin is not a particularly unusual event in corynebacteria.
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Details
- Title
- Bacterial sarcosine oxidase
- Creators
- Kalla L. Kvalnes-Krick
- Awarding Institution
- Hahnemann University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Hahnemann University; Philadelphia, Pennsylvania
- Number of pages
- ix, 139 pages, 29 unnumbered pages of plates
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Medicine (1982-1993); Hahnemann University (1982-1993); Biochemistry [Historical]
- Other Identifier
- 991021889011804721