Alcohol dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecane and hexadecanol metabolism

Abstract

Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH) exhibiting an apparent Km for ethanol of 512 microM and a Vmax of 138 nmol/min. An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme which exhibited an apparent Km for acetaldehyde of 50 microM and a Vmax of 183 nmol/min. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is a soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH exhibited apparent Kms for hexadecanol of 1.6 and 2.8 microM in crude extracts derived from hexadecane- and hexadecanol-grown cells, respectively. HDH was distinct from ADH-A and ADH-B, since HDH and ADH-A were not coinduced; Eth1 had wild-type levels of HDH; and HDH requires NAD, while ADH-B requires NADP. NAD- and NADP-independent HDH activity was not detected in the soluble or membrane fraction of extracts derived from hexadecane- or hexadecanol-grown cells. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.

Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanol metabolism

“…Induction experiments were performed as described previously (14), with palmitate-grown cells exposed to 0.3% (vol/vol) hexadecane, 0.2% (wt/vol) hexadecanol, 0.3% (vol/vol) ethanol, or 0.2% (vol/vol) dodecyl aldehyde.…”

“…Acinetobacter sp. strain HO1-N contains a soluble, NADlinked, ethanol-inducible acetylaldehyde dehydrogenase (ALDH) required for growth on ethanol (14). In this study, two distinct fatty aldehyde dehydrogenase (FALDH) activities were demonstrated in Acinetobacter sp.…”

“…strain HO1-N was used exclusively in this study (12). Culture conditions are described in the accompanying paper (14).…”

“…Isolation of fatty aldehyde-negative mutants. Cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and were subjected to two cycles of cycloserine-ampicillin enrichment during growth on minimal medium supplemented with 0.2% (vol/vol) dodecyl aldehyde (Aldrich Chemical Co., Inc., Milwaukee, Wis.), as described in the accompanying paper (14). After the second enrichment cycle, cells were screened for growth on minimal medium agar plates supplemented with dodecyl aldehyde, which was supplied as a vapor from the liquid phase in the lid of the petri dish.…”

“…Preparation of cell extracts and cell fractionation. Cell lysis and fractionation procedures have been described previously (14).…”