Acyl-CoA Dehydrogenases : characterization of the New Member Isobutyryl-CoA Dehydrogenase, Genetic Defects and Correlation to Thermal Unfolding

Abstract

Acyl-CoA dehydrogenases (ACADs) form a family of nine members that catalyze the ?-oxidation of acyl-CoA substrates, they require electron transfer flavoprotein (ETF) as electron acceptor, and differ in their specificity for different types of the fatty acids linked to CoA. The ACADs can be subdivided into two subfamilies. The first comprises five members (VLCAD1, VLCAD2, LCAD, MCAD and SCAD) that catalyze the ?-oxidation of straight chain substrates, which are degraded sequentially in the ß-oxidation cycle. The second subfamily includes four members (i3VD, i2VD, GCD and ACAD-8), which are involved in the degradation of fatty acid conjugates arising from amino acid catabolism.In my work I have studied a previously uncharacterized protein with ACAD-like sequence (ACAD-8) and have defined its substrate specificity. The enzyme is an isobutyryl-CoA dehydrogenase for which the abbreviation iBD has been introduced. A single patient has previously been identified whose fibroblasts exhibit a specific defect in valine metabolism. Amplified ACAD-8 cDNA made from patient fibroblast mRNA has a single nucleotide change (905G>A) in the ACAD-8 coding region compared to the sequence from ACAD-8 control cells. This encodes an Arg302Gln substitution in the full-length protein (position 280 in the mature protein), a position predicted by molecular modeling to be important in subunit interactions. The mutant enzyme was stable but was inactive when expressed in E. coli. It was also stable and appropriately targeted to mitochondria, but was inactive when expressed in mammalian cells.iBD was overexpressed in E. coli and purified to apparent homogeneity over various chromatographic steps. Recombinant iBD is a tetramer of four subunits of molecular weight ?42 KDa. It has a pI ?6.2, and a maximum activity with isobutyryl-CoA (100%, Km ?2.6 ?M), 15% with propionyl-CoA and very low activity with 2-methyl-butyryl-CoA. for isobutyryl-CoA. No activity was detected with n-butyryl-CoA and isovaleryl-CoA. iBD has sensitive SH group(s), modification of which affects the activity. The activity pattern is consistent with the proposed role of iBD in valine degradation.In view of the importance of ACAD enzymes and the occurrence of genetic defects that affect stability and activity, the thermal stability of both MCAD and i3VD and of relevant mutants was investigated. The study included also the effect substrates (octanoyl-CoA and isovaleryl-CoA for MCAD and i3VD, respectively) or substrate analogues (2-azaoctanoyl-CoA and 2-azaisovaleryl-CoA for MCAD and i3VD, respectively). The K304E- and T168A-MCAD mutants that occur in genetic defects, were also studied along with A282V-i3VD mutant. The midpoint transition, Tm, for MCAD wt, K304E and T168A that reflect the protein melting point were 53.6, 52.6 and 47 0C, respectively, in absence of ligand. In the presence of 20 µM of octanoyl-CoA, the values were 59.7, 58.3 and 53 0C, respectively. And in the presence of 300 µM of octanoyl-CoA, the values were 63.4, 61.6 and 56 0C, respectively. With the substrate analogue, 2-azaoctanoyl-CoA, Tm for MCAD wt, K304E and T168A were 57.5, 57.3 and 49.8 0C, respectively.