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

Lipoamide dehydrogenase (LPD) is an essential component of the pyruvate deh ydrogenase and 2-oxoglutarate dehydrogenase complexes, both playing a cruci al role within the central metabolism of aerobic organisms. Using oligonucl eotides designed according to conserved regions of LPD amino acid sequences from several organisms, the Ipd gene from Corynebacterium glutamicum was i dentified and subsequently subcloned. The cloned Ipd gene expressed in C. g lutamicum cells harbouring the gene on a plasmid showed a 12-fold higher sp ecific LPD activity when compared to the wild-type strain. DNA sequence ana lysis of a 4524 bp segment containing the Ipd gene and adjacent regions rev ealed that the Ipd 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 sequen ce of this polypeptide shows between 26 and 58 % identity when compared to LPD enzymes from other organisms. Transcriptional analyses revealed that th e Ipd gene from C. glutamicum is monocistronic (1(.)45 kb mRNA) and that it s transcription is initiated exactly at the nucleotide defined as the trans lational start. LPD was purified and biochemically characterized. This anal ysis revealed that the enzyme catalyses the reversible reoxidation of dihyd rolipoic acid and NADH:NAD(+) transhydrogenation, and is able to transfer e lectrons from NADH to various redox-active compounds and quinones. An in vi vo participation of C. glutamicum LPD in facilitation of quinone redox cycl ing is proposed.