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.
(Less)
- author
- Håkansson, Anders P
LU
and Smith, Alexander W
- organization
- publishing date
- 2007-10-05
- type
- Contribution to journal
- publication status
- published
- subject
- 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
- in
- Journal of Biological Chemistry
- volume
- 282
- issue
- 40
- pages
- 10 pages
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- scopus:35748954619
- pmid:17690105
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.M703144200
- language
- English
- LU publication?
- yes
- id
- b7730942-6798-48d1-b4e8-9e003a806f95
- date added to LUP
- 2016-05-21 10:51:19
- date last changed
- 2024-01-04 04:16:54
@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}},
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}},
language = {{eng}},
month = {{10}},
number = {{40}},
pages = {{30--29521}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
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}},
doi = {{10.1074/jbc.M703144200}},
volume = {{282}},
year = {{2007}},
}