Purification and Characterization of Membrane-Bound Aldehyde Dehydrogenase from Acinetobacter calcoaceticus Grown on Long-Chain Alkanes
Acinetobacter calcoaceticus grown on alkanes synthesizes a membrane-bound aldehyde dehydrogenase which oxidizes aliphatic aldehydes with NADP+ to the corresponding fatty acids. This enzyme is induced by exogenous alkanes, alcohols and aldehydes and repressed by intermediates of central metabolic pathways. It was shown ultracytochemically that it is located exclusively in the membranes which envelop intracellular hydrocarbon inclusions. The enzymic activity depends on cardiolipin.
Cytoplasma-free membranes were prepared by differential centrifugation after vibration with glass beads or lysozyme treatment of the microorganisms harvested in the log-phase. The enzyme was solubilized by detergents, purified in micellar form by chromatography on DEAE-cellulose and gel filtration on Sepharose CL-4B resulting in a 60fold enrichment. The enzyme is a tetrameric membrane protein with a molecular weight of 280 000 daltons. Its monomeric form is enzymatically inactive. The aldehyde dehydrogenase oxidizes homologous aliphatic aldehydes with differing efficiency. The apparent KM-values decrease with increasing chain length. At high substrate concentrations inhibition is observed. The aldehyde substrates are bound by hydrophobic interactions to the enzyme. Inhibition studies point to a functional important metal ion and an SH-group on the enzyme.
The enzyme can be incorporated into liposomal membranes prepared from bacterial lipids without loss of activity. Long-chain aldehyde substrates do not destroy the lipid vesicles. The reconstituted liposomal enzyme is very similar to its physiological form in the bacterial membrane as evidenced by an analogous behavior towards the action of proteases, phospholipases and detergents.
KeywordsAldehyde Dehydrogenase Aliphatic Aldehyde Acinetobacter Calcoaceticus Nitro Blue Tetrazolium Chloride Bacterial Lipid
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- Aurich H (1979): Die Oxydation aliphatischer Kohlenwasserstoffe durch Bakterien. Sitzungsber. Akad. Wiss. DDR 16 N, Akad.-Verl. Berlin, pp. 1–24Google Scholar
- Fennewald MS, Benson M, Oppici M, Shapiro J (1979): Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon. J. Bact. 139: 940–952Google Scholar
- Koelsch R, Lasch J, Klibanov AL, Torchilin VP (1981): Incorporation of chemically modified proteins into liposomes. Acta biol. med. germ. 40: 331–335Google Scholar
- Peterson JA, Basu D, Coon MJ (1966): Enzymatic co-oxidation. I. Electron carriers in fatty acid and hydrocarbon hydroxylation. J. Biol. Chem. 241: 5162–5164Google Scholar
- Rothe U, Schöpp W, Aurich H (1976): Enzymatischer Umsatz von Tetradekanol in heterogener Phase durch Hefe-Alkoholdehydrogenase. Acta biol. med. germ. 35: 7–14Google Scholar
- Schöpp W, Aurich H (1973): Abhängigkeit der KM- und Vmax-Werte von der Kettenlänge des Substrates für die Reaktion der Alkoholdehydrogenase aus Hefe. Acta biol. med. germ. 31: 19–28Google Scholar
- Scott CCL, Finnerty RW (1976): Characterization of intracytoplasmic hydrocarbon inclusions from the hydrocarbon-oxidizing Acinetobacter species H01-N. J. Bact. 127: 481–489Google Scholar
- Sorger H, Aurich H (1978): Mikrobielle Aldehyddehydrogenasen und ihre Bedeutung für die Assimilation aliphatischer Kohlenwasserstoffe. Wiss. Z. KMU 27: 35–46Google Scholar
- Tauchert H, Schöpp W, Aurich H (1978): Pyridinnukleotid-unabhängige Alkoholdehydrogenase in alkanverwertenden Bakterien. Wiss. Z. KMU 27: 25–34Google Scholar
- Vořišek J, Sorger H, Aurich H, Lojda Z (1982): Ultracytochemical staining of aldehyde dehydrogenase activity in Acinetobacter calcoaceticus cultivated on n-alkanes. Symp. histochem. Karlovy VaryGoogle Scholar