Activity of Human Dihydrolipoamide Dehydrogenase Is Reduced by Mutation at Threonine-44 of FAD-binding Region to Valine

Kim, Hakjung

doi:10.5483/bmbrep.2002.35.4.437

Cited by 3 publications

(3 citation statements)

References 10 publications

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“…According to the sequence of 1jehB, a disulfide bond is formed between Cys44 and Cys49, which are essential for the dehydrogenase activity [13]. The disulfide bond at the active site of E3 of various species should be broken during the catalysis to form an intermediate with the substrate [17]. Thus there is no such a similar active disulfide bond located in the sequence of 2Q7V, suggesting that this disulfide bond is not necessary for DLDH.…”

Section: Homology Modelingmentioning

confidence: 99%

Structural Characterization of FAD-binding Domain of CglE, a Putative Dehydrolipoamide Dehydrogenase in Meningitic E. coli K1

Wang¹

2013

J Data Mining Genomics Proteomics

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To characterize the structural features of FAD-binding domain of E. coli K1 CglE, a dehydrolipoamide dehydrogenase (DLDH) by homology modeling. Sequence similarity of N-terminal residues 1-70 with the a-subunit of FAD-binding domain from CglE of E. coli K1 and other DLDHs provided a basis for the design of the FAD-binding domain of CglE. As a result of finding no single satisfied template for the homology modeling for CglE, two templates (PDB code 2q7vA and 1jehA) were obtained by an online homology modeling procedure for multi-templates modeling. To obtain a high quality target protein, a computational bioinformatic software Accelrys Discovery Studio client 2.5 and several automated online servers were utilized. Due to the relatively low identity of the alignments, two templates were taken into consideration attempting to improve the homology model. The quality of the refined model was assessed on the basis of both geometric and energetic aspects including MD simulations, energy minimizations, Ramachandran Plot and other measurements.

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Section: Homology Modelingmentioning

confidence: 99%

Structural Characterization of FAD-binding Domain of CglE, a Putative Dehydrolipoamide Dehydrogenase in Meningitic E. coli K1

Wang¹

2013

J Data Mining Genomics Proteomics

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“…In addition, nine artificial substitution mutations were created and characterized. Among them, three were located at FAD-binding domain [23][24][25], one was located at NAD + -binding domain [23], three were located at interface domain [26,27], and two were located at center domain [28]. The characterization of these mutants leads the way to understand the structure-function relationship of E3.…”

Section: Introductionmentioning

confidence: 98%

The role of amino acids T148 and R281 in human dihydrolipoamide dehydrogenase

Wang

et al. 2007

J Biomed Sci

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Human dihydrolipoamide dehydrogenase (hE3) is a common component of alpha-ketoacid dehydrogenase complexes. Mutations of this homodimeric protein cause E3 deficiency and are always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other 17 eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, two amino acids, T148 and R281, were subjected to mutagenesis and four hE3 mutants, T148G, T148S, R281N, and R281K, were expressed and assayed. The specific activities of T148G, T148S, R281N, and R281K are 76.34%, 88.62%, 12.50%, and 11.93% to that of wild-type E3, respectively. The FAD content analysis indicated that the FAD content of these mutant E3s were about 71.0%, 92%, 96%, and 93% that of wild-type E3, respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic data demonstrated that the K(cat) of forward reaction of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and R281 play a role in the catalytic function of the E3.

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“…The recombinant human E3 has possessed similar properties to those of purified mammalian E3s. The putative essential amino acid residues of human E3 have been modified using site-directed mutagenesis and the mutants have been characterized (Kim and Patel, 1992;Leu et al, 1995;Kim, 2002).…”

Section: Introductionmentioning

confidence: 99%

Asparagine-473 Residue Is Important to the Efficient Function of Human Dihydrolipoamide Dehydrogenase

Kim¹

2005

BMB Reports

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Dihydrolipoamide dehydrogenase (E3) catalyzes the reoxidation of dihydrolipoyl moiety of the acyltransferase components of three alpha-keto acid dehydrogenase complexes and of the hydrogen-carrier protein of the glycine cleavage system. His-457 of Pseudomonas putida E3 is suggested to interact with the hydroxyl group of Tyr-18 of the other subunit and with Glu-446, a component in the last helical structure. To examine the importance of the suggested interactions in human E3 function, the corresponding residue of human E3, Asn-473, was substituted to Leu using site-directed mutagenesis. The E3 mutant was expressed in Escherichia coli and highly purified using an affinity column. Its E3 activity was decreased about 37-fold, indicating that Asn-473 residue was important to the efficient catalytic function of human E3. Its slightly altered spectroscopic properties implied that small conformational changes could occur in the E3 mutant.

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Activity of Human Dihydrolipoamide Dehydrogenase Is Reduced by Mutation at Threonine-44 of FAD-binding Region to Valine

Cited by 3 publications

References 10 publications

Structural Characterization of FAD-binding Domain of CglE, a Putative Dehydrolipoamide Dehydrogenase in Meningitic E. coli K1

Structural Characterization of FAD-binding Domain of CglE, a Putative Dehydrolipoamide Dehydrogenase in Meningitic E. coli K1

The role of amino acids T148 and R281 in human dihydrolipoamide dehydrogenase

Asparagine-473 Residue Is Important to the Efficient Function of Human Dihydrolipoamide Dehydrogenase

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