Identification and Characterization of the acoD Gene Encoding a Dihydrolipoamide Dehydrogenase of the Klebsiella pneumoniae Acetoin Dehydrogenase System

Peng, Hwei-Ling; Deng, Wen-Ling; Yang, Yin Hsiu; Chang, Hwan‐You

doi:10.1093/oxfordjournals.jbchem.a021357

Cited by 3 publications

(2 citation statements)

References 15 publications

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“…The lpd gene encodes a protein of 466 amino acids with a calculated mass of 49.8 kDa. The predicted amino acid sequence was closely related to other lipoamide dehydrogenases with up to 54% identity to the E3 component of the acetoin dehydrogenase complex of Klebsiella pneumoniae (46) and 40% identity to the Lpd protein of Pseudomonas fluorescens (5). The sequence contained the characteristic motifs of flavincontaining disulfide oxidoreductases (71) (Fig.…”

Section: Resultsmentioning

confidence: 94%

Purification of the Pyruvate Dehydrogenase Multienzyme Complex of Zymomonas mobilis and Identification and Sequence Analysis of the Corresponding Genes

et al. 1998

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The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1α (38 kDa) and E1β (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome ofZ. mobilis. The genes pdhAα (1,065 bp) andpdhAβ (1,389 bp), encoding the E1α and E1β subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAα,pdhAβ, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of thepdhB gene contains a single lipoyl domain. The E1β subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.

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Section: Resultsmentioning

confidence: 94%

Purification of the Pyruvate Dehydrogenase Multienzyme Complex of Zymomonas mobilis and Identification and Sequence Analysis of the Corresponding Genes

et al. 1998

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“…In Alcaligenes eutrophus, the structural genes identified for E1␣ (acoA), E1␤ (acoB), and E2 (acoC) are clustered with acoX, which encodes a protein of unknown function in the order of acoXABC (28). The structural gene for E3 which is not found in A. eutrophus aco operon, however, is present as acoL in Pelobacter carbinolicus acoABCLS (25) and Clostridium magnum acoABXCL (19) operons and as acoD in the Klebsiella pneumoniae acoABCD operon (27). Amino acid sequences deduced from these genes exhibit significant homology to those of the respective component of various 2-oxo acid dehydrogenase complexes, suggesting an evolutionary relationship among these catabolic systems.…”

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confidence: 99%

Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon

et al. 1997

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By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli. The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon. This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E. coli maltose regulon. A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified. Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK. Several copies of NtrC recognition sequence to N 18 )-GTG] were found in the promoter regions of both the acoK gene and the acoABCD operon. Acetoin-dependent expression of the acoABCD operon could be restored in the E. coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product. The AcoK protein overproduced in E. coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence. A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated.Many bacteria are able to utilize acetoin as a carbon source. In some of the bacteria, dissimilation of acetoin proceeds via an oxidative cleavage process which is catalyzed by an acetoin dehydrogenase enzyme system with acetaldehyde and acetyl coenzyme A as the end products (13, 22). The acetoin dehydrogenase enzyme system is composed of E1␣ and E1␤, an acetoin-dependent dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR); E2, a dihydrolipoamide acetyltransferase; and E3, a dihydrolipoamide dehydrogenase. Structural genes encoding E1␣, E1␤, E2, and E3 of the acetoin dehydrogenase enzyme system have recently been isolated from several bacterial species (11,19,25,28). In Alcaligenes eutrophus, the structural genes identified for E1␣ (acoA), E1␤ (acoB), and E2 (acoC) are clustered with acoX, which encodes a protein of unknown function in the order of acoXABC (28). The structural gene for E3 which is not found in A. eutrophus aco operon, however, is present as acoL in Pelobacter carbinolicus acoABCLS (25) and Clostridium magnum acoABXCL (19) operons and as acoD in the Klebsiella pneumoniae acoABCD operon (27). Amino acid sequences deduced from these genes exhibit significant homology to those of the respective component of various 2-oxo acid dehydrogenase complexes, suggesting an evolutionary relationship among these catabolic systems.Despite the wealth of information on acetoin dehydrogenases, how these aco operons are regulated is less understood. The acoR gene located upst...

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The Disruption of a Gene Encoding a Putative Arylesterase Impairs Pyruvate Dehydrogenase Complex Activity and Nitrogen Fixation in Sinorhizobium meliloti

Soto¹,

Sanjuán²,

Olivares³

2001

MPMI

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Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the trichloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3' end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.

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Identification and Characterization of the acoD Gene Encoding a Dihydrolipoamide Dehydrogenase of the Klebsiella pneumoniae Acetoin Dehydrogenase System

Cited by 3 publications

References 15 publications

Purification of the Pyruvate Dehydrogenase Multienzyme Complex of Zymomonas mobilis and Identification and Sequence Analysis of the Corresponding Genes

Purification of the Pyruvate Dehydrogenase Multienzyme Complex of Zymomonas mobilis and Identification and Sequence Analysis of the Corresponding Genes

Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon

The Disruption of a Gene Encoding a Putative Arylesterase Impairs Pyruvate Dehydrogenase Complex Activity and Nitrogen Fixation in Sinorhizobium meliloti

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