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

Abstract

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.

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

“…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.…”