Catalytic and FAD-binding Residues of Mitochondrial Very Long Chain Acyl-Coenzyme A Dehydrogenase

Masayoshi Souri,Toshifumi Aoyama,Gerald F Cox,Takashi Hashimoto

doi:10.1074/jbc.273.7.4227

Masayoshi Souri, Toshifumi Aoyama + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.7.4227

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Abstract

Very long-chain acyl-CoA dehydrogenase (VLCAD) is one of four flavoproteins which catalyze the initial step of the mitochondrial beta-oxidation spiral. By sequence comparison with other acyl-CoA dehydrogenases, Glu-422 of VLCAD has been presumed to be the catalytic residue that abstracts the alpha-proton in the alphabeta-dehydrogenation reaction. Replacing Glu-422 with glutamine (E422Q) caused a loss of enzyme activity by preventing the formation of a charge transfer complex between VLCAD and palmitoyl-CoA. This result provides further evidence for Glu-422 being part of the active site of VLCAD. F418L is a disease-causing mutation in human VLCAD deficiency. Unlike wild-type VLCAD, F418L and F418V contained no bound FAD when expressed at extremely high levels in the baculovirus expression system. Although F418T and F418Y bound FAD at a level similar to that of wild-type VLCAD, both showed reduced Vmax values toward palmitoyl-CoA, most likely due to a decrease in the rate of enzyme-bound FAD reduction. These data suggest that Phe-418 is involved in the binding and subsequent reduction of FAD. FAD-deficient VLCADs (F418L, F418V, and apo-VLCAD) showed increased sensitivity to trypsinization. Loss of FAD may change the folding of VLCAD subunit.

Highlights

F418L is a disease-causing mutation in human Very longchain acyl-CoA dehydrogenase (VLCAD) deficiency
Two subfamilies of acyl-CoA dehydrogenases may be distinguished by the position of a catalytic glutamate residue: one subfamily corresponds to Glu-376 of MCAD, and the other corresponds to Glu-261 of LCAD [12, 13, 15]
Sequence alignments suggest that SCAD and VLCAD belong to the MCAD subfamily (Glu-376), while IVD belongs to the LCAD subfamily (Glu-261) (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Materials—VLCAD was purified from human liver as described [2]. Human VLCAD cDNA was cloned previously [9]. Site-directed mutagenesis was performed by the method of Deng and Nickoloff [25] using a TransformerTM Site-Directed Mutagenesis Kit. Mutant cDNAs were synthesized according to the manufacturer’s instructions, using pT7B-VLCAD as template. Expressed protein was extracted from the mitochondrial fraction and purified by phosphocellulose and DEAE-cellulose column chromatography as described previously [1]. FAD Content—Fifty ␮l of the partially purified sample (approximately 50 ␮g) was diluted with 350 ␮l of 20 mM potassium phosphate (pH 7.5), and 25 ␮l of 50% trichloroacetate was added. Enzyme Assay—VLCAD enzyme activity was measured by the dyereduction method using PES as an electron transfer dye and dichloroindophenol as an electron acceptor. The reaction mixture contained 50 mM potassium phosphate (pH 7.5), 30 ␮M acyl-CoA, 35 ␮M dichloroindophenol, 1 mM N-ethylmaleimide, 3 mM PES, and enzyme. The activity was calculated using a molar extinction coefficient of 21,000 MϪ1 cmϪ1

RESULTS

Native molecular massa

DISCUSSION