Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

Abstract

The N-terminal amino acid sequence of purified acetaldehyde dehydrogenase II (AcDH-II) from ethanolgrown cells ofAlcaligenes eutrophus was determined. By using oligonucleotides deduced from this sequence the structural gene for AcDH-II, which was referred to as acoD, was localized on a 7.2-kbp EcoRI restriction fragment (fragment D), which has been cloned recently (C. Frund, H. Priefert, A. Steinbuchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989 The aerobic bacterium Alcaligenes eutrophus H16 synthesizes two different NAD-dependent acetaldehyde dehydrogenases (AcDH-I and AcDH-II; EC 1.2.1.3) during growth on acetoin. Both dehydrogenases are also synthesized in the type strain TF93 and in ethanol-utilizing mutants of A. eutrophus derived from strains H16 or N9A during growth on ethanol or on acetoin. Since the expression of AcDH-II is specifically induced only during growth on these substrates, this enzyme seems to be responsible for the oxidation of acetaldehyde to acetate in both catabolic pathways. AcDH-II possesses a high affinity toward acetaldehyde (Km = 4 ,uM), exhibits a wide substrate spectrum, and amounts to about 14% of the total soluble protein in ethanol-grown cells (21).In A. eutrophus the degradation of acetoin is initiated by direct cleavage into two C2 compounds (13). This reaction is catalyzed by acetoin:2,6-dichlorophenolindophenol oxidoreductase and occurs also in Bacillus subtilis (28) and in the anaerobic bacterium Pelobacter carbinolicus (33) during growth on acetoin. Recently, we have identified and localized the genes, which are essentially involved in the catabolism of acetoin in A. eutrophus, on five different genomic EcoRI restriction fragments (A, B, C, D, and E) (13). The structural genes for the a-and P-subunits of acetoin:2,6-dichlorophenolindophenol oxidoreductase (acoA and acoB, respectively); for the protein FMP (acoC), which seems to be a dihydrolipoamide acetyltransferase; and for a protein of yet unknown function (acoX) are probably organized in one single operon (acoXABC) which is localized on fragments A and C (36). Fragment B harbors an rpoN-like gene, and mutants carrying a TnS insertion in this gene exhibited an extremely pleiotropic phenotype (13,38). As these mutants * Corresponding author.were also unable to utilize acetoin as a sole carbon source for growth, the expression of acetoin-catabolic genes in A. eutrophus seems to be also rpoN dependent. This is consistent with the identification of a promoter exhibiting striking homology to the enterobacterial c54-dependent promoter consensus sequence upstream of acoX-ABC. The information encoded by fragments D and E remained to be elucidated.In the present study we localized the structural gene for AcDH-II (acoD), which is in A. eutrophus essentially involved in the catabolism of acetoin as well as of ethanol, on EcoRI restriction fragment D. The results of the molecular characterization of acoD and of adjacent DNA regions confirmed our assumption that the expression of acoD is also rpoN dependent, like the ex...

aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp

“…4). Synthesis of the related aldehyde dehydrogenases AcoD from A. eutrophus and AldA from V. cholerae has been reported to be dependent on RpoN and ToxR (a transcriptional activator), respectively (41,42). We did not observe any effect on aldB-731 LacZ levels by an rpoN mutation (57).…”

“…The highest level of expression among members of this latter group occurs during early stationary phase. On the basis of the high degree of identity between the partial amino acid sequence of AldB deduced from the DNA sequence at the junction of the lac fusion and that of an NAD-dependent acetaldehyde dehydrogenase encoded by the acoD gene of A. eutrophus (42), we proposed that aldB encodes an aldehyde dehydrogenase in E. coli (56). The potential functions of other Fis-repressed and RpoS-activated genes that were identified are not yet known.…”

“…Enzymes within this group may play roles in multiple pathways in cellular metabolism (4). Many of the identified aldehyde dehydrogenases, including those from bacteria, fungi, plants, and mammalian cells, share significant similarities in their primary structures (15,20,42,54).…”

“…Some aldehyde dehydrogenases, such as the malonic semialdehyde dehydrogenase of Pseudomonas aeruginosa (34), have high specificity, while others function on a broad spectrum of substrates. Two examples of this latter group are the aldehyde dehydrogenase encoded by the ald gene of Escherichia coli (20) and the acetaldehyde dehydrogenase II encoded by the acoD gene of Alcaligenes eutrophus (42). Enzymes within this group may play roles in multiple pathways in cellular metabolism (4).…”

A proteomic study of Methylobacterium extorquens reveals a response regulator essential for epiphytic growth

“…Of these, RMQ03452 is phyllosphere-specific, sharing a high percentage of sequence identity with the AcoD of Ralstonia eutrophus, which is involved in the catabolism of acetoin and ethanol (20), and with AldB of Escherichia coli, which is thought to have a role in detoxifying alcohols and aldehydes (21). The RMQ03452 protein in M. extorquens might contribute to carbon dissimilation or detoxification of alcohols or aldehydes that are produced by plants (22).…”