Enzymatic characterization of dihydrolipoamide dehydrogenase from Streptococcus pneumoniae harboring its own substrate

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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.

Details

Authors
Organisations
External organisations
  • University at Buffalo
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Infectious Medicine
  • Cell and Molecular Biology

Keywords

  • Dihydrolipoamide Dehydrogenase, Flavins, Kinetics, Models, Chemical, NAD, Protein Binding, Protein Structure, Tertiary, Pyruvate Dehydrogenase Complex, Recombinant Proteins, Sequence Analysis, DNA, Streptococcus pneumoniae, Substrate Specificity, Sulfhydryl Compounds, Thioctic Acid
Original languageEnglish
Pages (from-to)29521-30
Number of pages10
JournalJournal of Biological Chemistry
Volume282
Issue number40
Publication statusPublished - 2007 Oct 5
Publication categoryResearch
Peer-reviewedYes