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

Institute of Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany¹
Institut de Génétique et Microbiologie, Bat. 360, Université Paris-Sud, Centre d’Orsay, F-91405 Orsay Cedex, France²

Author for correspondence: Armel Guyonvarch. Tel: +33 1 69 15 63 41. Fax: +33 1 69 15 63 34. e-mail: armel{at}igmors.u-psud.fr

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 lpd gene from Corynebacterium glutamicum was identified and subsequently subcloned. The cloned lpd gene expressed in C. glutamicum 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 lpd gene and adjacent regions revealed that the lpd 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 M_r 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 lpd gene from C. glutamicum is monocistronic (1·45 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 in vivo participation of C. glutamicum LPD in facilitation of quinone redox cycling is proposed.

Keywords: Corynebacterium glutamicum, lpd gene, initiation of transcription, initiation of translation

Abbreviations: DCIP, 2,6-dichlorophenolindophenol; INT, 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride; LPD, lipoamide dehydrogenase

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

^a Present address: Instituto de Ciencia e Tecnologia dos Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

^b Present address: University of Ulm, Department of Microbiology and Biotechnology, D-89069 Ulm, Germany.

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