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

Håkansson, Anders P LU and Smith, Alexander W (2007) In Journal of Biological Chemistry 282(40). p.30-29521
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... (More)

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.

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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
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Journal of Biological Chemistry
volume
282
issue
40
pages
10 pages
publisher
ASBMB
external identifiers
  • scopus:35748954619
ISSN
0021-9258
DOI
10.1074/jbc.M703144200
language
English
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yes
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b7730942-6798-48d1-b4e8-9e003a806f95
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2016-05-21 10:51:19
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2017-01-01 08:26:45
@article{b7730942-6798-48d1-b4e8-9e003a806f95,
  abstract     = {<p>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.</p>},
  author       = {Håkansson, Anders P and Smith, Alexander W},
  issn         = {0021-9258},
  keyword      = {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},
  language     = {eng},
  month        = {10},
  number       = {40},
  pages        = {30--29521},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Enzymatic characterization of dihydrolipoamide dehydrogenase from Streptococcus pneumoniae harboring its own substrate},
  url          = {http://dx.doi.org/10.1074/jbc.M703144200},
  volume       = {282},
  year         = {2007},
}