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.
KeywordsHydrocarbon Assimilation Pseudomonas NADPH NADH
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