title: Enzymatic characterization of dihydrolipoamide dehydrogenase from Streptococcus pneumoniae harboring its own substrate.
abstract: This study describes the enzymatic characterization of dihydrolipoamide dehydrogenase (DLDH) from Streptococcus pneumoniae and is the first characterization of a DLDH that carries its own substrate (a lipoic acid covalently attached to a lipoyl protein domain) within its own sequence. Full-length recombinant DLDH (rDLDH) was expressed and compared with enzyme expressed in the absence of lipoic acid (rDLDH(-LA)) or with enzyme lacking the first 112 amino acids constituting the lipoyl protein domain (rDLDH(-LIPOYL)). All three proteins contained 1 mol of FAD/mol of protein, had a higher activity for the conversion of NAD(+) to NADH than for the reaction in the reverse direction, and were unable to use NADP(+) and NADPH as substrates. The enzymes had similar substrate specificities, with the K(m) for NAD(+) being approximately 20 times higher than that for dihydrolipoamide. The kinetic pattern suggested a Ping Pong Bi Bi mechanism, which was verified by product inhibition studies. The protein expressed without lipoic acid was indistinguishable from the wild-type protein in all analyses. On the other hand, the protein without a lipoyl protein domain had a 2-3-fold higher turnover number, a lower K(I) for NADH, and a higher K(I) for lipoamide compared with the other two enzymes. The results suggest that the lipoyl protein domain (but not lipoic acid alone) plays a regulatory role in the enzymatic characteristics of pneumococcal DLDH.
authors: Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, New York 14214, USA. andersh@buffalo.edu
authors: Anders P
authors: AP
authors: Håkansson
authors: Alexander W
authors: AW
authors: Smith
mesh_terms: D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D008063:Thioctic Acid / Q000737:chemistry
chemical_list: D005415:Flavins; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D009243:NAD; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase