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

Abstract

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.

The vignette for V15 N1 issue

“…The four serotypes of Dengue viruses cause the most important arboviral disease in the tropical and subtropical areas. This study [5] indicates that R281 is essential for E3 catalytic function but not T148. Subgenomic replicons, which contain all the non-structural proteins required for amplifying themselves but lack some or all of the structural proteins, are useful tools to study viral replication in the absence of virion assembly and maturation [2].…”