Characterization of Two Naturally Occurring Mutations Close to Cofactors in Human Dihydrolipoamide Dehydrogenase

Abstract

Dihydrolipoamide dehydrogenase (E3) (dihydrolipoamide: NAD oxidoreductase; EC 1.8.1.4) exists as a common component in three α-keto acid dehydrogenase complexes (pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenase complexes) and the glycine cleavage system. It catalyzes the reoxidation of the dihydrolipoyl group of the acyltransferase components of the α-keto acid dehydrogenase complexes and the reoxidation of that of the hydrogen-carrier protein of the glycine cleavage system. It is a homodimeric enzyme containing one FAD as a prosthetic group at each subunit. Two naturally occurring mutations near cofactors in human E3 were reported. One mutation was from E3 deficient patients of Ashkenazi Jewish. A common mutation of Gly-194 to Cys was found in their E3 sequences. Their clinical symptoms were the recurrent attacks of vomiting, abdominal pain, neonatal suffering, muscle weakness and exertional fatigue. Another mutation in a boy having reduced E3 activity was also reported. At the age of ten weeks, his clinical symptoms were vomiting, progressive hypotonia and a severe lactic acidosis. He possessed a mutation of Met-326 to Val in his E3 sequence. Site-directed mutagenesis method has been a useful tool for the structure-function study of human E3 and other proteins. By the site-specific mutations of Gly-194 to Cys and Met-326 to Val, the effects of these naturally occurring mutations close to cofactors in human E3 structure and function were examined. Site-directed mutagenesis was performed using a mutagenesis kit according to the provided protocol. PCRs were carried out using the human E3 expression vector pPROEX-1:E3 as a template in a programmable PCR machine with the appropriate primers. Whole DNA sequence of human E3 coding region was sequenced to verify the integrity of DNA sequences and the mutations. Purification of the Val-326 mutant was unsuccessful. There was not enough of the Val-326 mutant present to be purified when its expression was induced in E. coli. The SDS-PAGE gel showed that very small amounts of the mutant were present in the soluble fraction. This indicated that the mutant could have problems in its structural stability and/or proper folding processes while it was expressed in E. coli. The Met-326 is located very close to both FAD and NAD in the E3 structure as shown in Figure 1A and involved in several direct interactions with them. The substitution of the Met-326 with smaller Val probably causes structural changes that affect more directly the bindings of near FAD and NAD to the enzyme (Figure 1B). These structural changes could be detrimental enough to destroy the stable expression of the enzyme in E. coli. The Cys-194 mutant was expressed in E. coli enough to be purified and characterized even though the amounts of purified enzyme were about 12-fold lesser than those of normal enzyme. The purification of the Cys-194 mutant was performed using nickel affinity column according to the provided protocol. SDS-PAGE gel showed that the mutant was highly purified. E3 assay was performed at 37 C in a 50 mM potassium phosphate buffer (pH 8.0) containing 1.5