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Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in
P Wallbrandt1, V Tegman, B H Jonsson
1Department of Biochemistry, University of Umeå, Sweden.
Abstract:
A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex.
Insights
Researchers identified four genes in Acholeplasma laidlawii encoding proteins homologous to the pyruvate dehydrogenase complex. These genes, found on a plasmid, restored enzyme activity in E. coli mutants, suggesting they code for this essential metabolic complex.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Acholeplasma laidlawii is a bacterium lacking a cell wall.
- The pyruvate dehydrogenase complex is a crucial enzyme complex in cellular metabolism.
- Understanding the genetic basis of metabolic pathways in A. laidlawii is important for comparative genomics.
Purpose of the Study:
- To identify and characterize genes encoding the pyruvate dehydrogenase complex in Acholeplasma laidlawii.
- To determine the function of these genes through complementation studies.
- To investigate the localization and activity of the encoded proteins.
Main Methods:
- Genomic library screening using antibodies.
- Nucleotide sequencing of a plasmid insert.
- Amino acid sequencing for protein terminus verification.
- Homology searches using computer-aided methods.
- Immunological detection of proteins.
- Enzyme activity assays in vivo complementation experiments.
Main Results:
- A 4.6-kbp plasmid insert containing four sequential genes (coding for 39, 36, 57, and 36 kDa proteins) was identified.
- Translated DNA sequences showed homology to pyruvate dehydrogenase complex subenzymes.
- The plasmid-encoded E2 protein was recognized by antibodies against Bacillus subtilis E2 subenzymes.
- Pyruvate dehydrogenase enzyme activity was restored in E. coli mutants complemented with the A. laidlawii genes.
- A significant portion of the E2 protein and pyruvate dehydrogenase activity were associated with the cytoplasmic membrane.
Conclusions:
- The sequenced genes on the plasmid likely code for the pyruvate dehydrogenase complex in Acholeplasma laidlawii.
- The identified proteins are functional and can restore pyruvate dehydrogenase activity in a heterologous system.
- The association of the E2 protein and activity with the cytoplasmic membrane suggests a specific localization.