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Volume 147, Issue 8

Lipoamide dehydrogenase from : molecular and physiological analysis of the gene and characterization of the enzyme

The GenBank accession number for the nucleotide sequence determined in this work is Y16642.

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Abstract

Lipoamide dehydrogenase (LPD) is an essential component of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, both playing a crucial role within the central metabolism of aerobic organisms. Using oligonucleotides designed according to conserved regions of LPD amino acid sequences from several organisms, the gene from was identified and subsequently subcloned. The cloned gene expressed in cells harbouring the gene on a plasmid showed a 12-fold higher specific LPD activity when compared to the wild-type strain. DNA sequence analysis of a 4524 bp segment containing the gene and adjacent regions revealed that the gene is not flanked by genes encoding other subunits of the pyruvate or 2-oxoglutarate dehydrogenase complexes and predicted an LPD polypeptide of 469 amino acids with an of 50619. The amino acid sequence of this polypeptide shows between 26 and 58% identity when compared to LPD enzymes from other organisms. Transcriptional analyses revealed that the gene from is monocistronic (145 kb mRNA) and that its transcription is initiated exactly at the nucleotide defined as the translational start. LPD was purified and biochemically characterized. This analysis revealed that the enzyme catalyses the reversible reoxidation of dihydrolipoic acid and NADH:NAD transhydrogenation, and is able to transfer electrons from NADH to various redox-active compounds and quinones. An participation of LPD in facilitation of quinone redox cycling is proposed.

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Keyword(s): Corynebacterium glutamicum , DCIP, 2,6-dichlorophenolindophenol , initiation of transcription , initiation of translation , INT, 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride , lpd gene and LPD, lipoamide dehydrogenase
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2001-08-01
2024-04-26
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Lipoamide dehydrogenase from Corynebacterium glutamicum: molecular and physiological analysis of the lpd gene and characterization of the enzymeThe GenBank accession number for the nucleotide sequence determined in this work is Y16642.
Microbiology 147, 2223 (2001); https://doi.org/10.1099/00221287-147-8-2223
/content/journal/micro/10.1099/00221287-147-8-2223
/content/journal/micro/10.1099/00221287-147-8-2223
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