Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway.

The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), apoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA, acoB, and acoC for E1 alpha (M(r) 34639), E1 beta (M(r) 37268), and E2 (M(r) 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M(r) 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.

Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system.

E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product, which is a regulatory protein required for expression of the A. eutrophus aco genes, and the A. eutrophus acoX gene product, which has an unknown function, respectively. The aco genes of C. magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.

Identification and molecular characterization of the aco genes encoding the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system. Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found. In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P. carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes.

Purification and characterization of the E1 component of the Clostridium magnum acetoin dehydrogenase enzyme system.

In Clostridium magnum strain Wo Bd P1 the formation of the enzyme components of the acetoin dehydrogenase enzyme system E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase Ao:DCPIP OR), E2 (dihydrolipoamide acetyltransferase DHLTA) and E3 (dihydrolipoamide dehydrogenase DHLDH) were induced during growth on acetoin. Ao:DCPIP OR was purified from acetoin-grown cells in two steps by chromatography on DEAE-Sephacel and on Mono Q HR. Native Ao:DCPIP OR exhibited a M(r) of 138,000; it consisted of two different subunits of M(r) alpha 38,500 and M(r) beta 34,000, and it occurred most probably in a tetrameric alpha 2 beta 2 structure. The N-terminal amino acid sequences of the alpha- and beta-subunits revealed homologies to the N-termini of the corresponding subunits of Ao:DCPIP OR from Pelobacter carbinolicus and from Alcaligenes eutrophus; furthermore, the N-terminus of the beta-subunit exhibited homologies to the N-termini of beta-subunits from different 2-oxo acid dehydrogenases.

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

Dihydrolipoamide dehydrogenase (DHLDH), dihydrolipoamide acetyltransferase (DHLTA), and acetoin: 2,6-dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) were purified from acetoin-grown cells of Pelobacter carbinolicus. DHLDH had a native Mr of 110,000, consisted of two identical subunits of Mr 54,000, and reacted only with NAD(H) as a coenzyme. The N-terminal amino acid sequence included the flavin adenine dinucleotide-binding site and exhibited a high degree of homology to other DHLDHs. DHLTA had a native Mr of greater than 500,000 and consisted of subunits identical in size (Mr 60,000). The enzyme was highly sensitive to proteolytic attack. During limited tryptic digestion, two major fragments of Mr 32,500 and 25,500 were formed. Ao:DCPIP OR consisted of two different subunits of Mr 37,500 and 38,500 and had a native Mr in the range of 143,000 to 177,000. In vitro in the presence of DCPIP, it catalyzed a thiamine pyrophosphate-dependent oxidative-hydrolytic cleavage of acetoin, methylacetoin, and diacetyl. The combination of purified Ao:DCPIP OR, DHLTA, and DHLDH in the presence of thiamine pyrophosphate and the substrate acetoin or methylacetoin resulted in a coenzyme A-dependent reduction of NAD. In the strictly anaerobic acetoin-utilizing bacteria P. carbinolicus, Pelobacter venetianus, Pelobacter acetylenicus, Pelobacter propionicus, Acetobacterium carbinolicum, and Clostridium magnum, the enzymes Ao:DCPIP OR, DHLTA, and DHLDH were induced during growth on acetoin, whereas they were absent or scarcely present in cells grown on a nonacetoinogenic substrate.

Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate.

Dihydrolipoamide dehydrogenase and dihydrolipoamide acetyltransferase were formed when Pelobacter carbinolicus strain GraBd1 was grown on acetoin. The specific activities of these enzymes amounted to 0.50 and 28.7 U/mg protein, respectively. The crude extract catalyzed the CoASH- and NAD+-dependent formation of acetyl-CoA from acetoin and methylacetoin. From ethylene glycol-grown cells these activities were absent. Crude extracts also exhibited acetoin: methyl viologen and acetoin: metronidazole oxidoreductase activity. As shown by reconstitution experiments methylviologen reduction was dependent on the presence of a light-brownish protein (Mr 220,000 +/- 10,000); metronidazole reduction was in addition dependent on the presence of a dark-brownish protein (Mr 4,900 +/- 800), which is probably a ferredoxin. However, both components were synthesized constitutively. We discussed a model for oxidative-thiolytic cleavage of acetoin which is analogous to the reaction of the pyruvate dehydrogenase enzyme complex rather than to pyruvate: ferredoxin oxidoreductase.