The Human Apoptosis-inducing Protein AMID Is an Oxidoreductase with a Modified Flavin Cofactor and DNA Binding Activity

• Published in: The Journal of biological chemistry 2005-09, Vol.280 (35), p.30735-30740
• Main Authors: Marshall, Ker R., Gong, Min, Wodke, Leigh, Lamb, John H., Jones, Donald J.L., Farmer, Peter B., Scrutton, Nigel S., Munro, Andrew W.
• Format: Article
• Language: English
• Subjects: Apoptosis - physiology; Apoptosis Regulatory Proteins; Coenzymes - chemistry; Coenzymes - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Flavin-Adenine Dinucleotide - analogs & derivatives; Flavin-Adenine Dinucleotide - metabolism; Flavins - chemistry; Flavins - metabolism; Humans; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Molecular Sequence Data; Molecular Structure; NAD - metabolism; NADP - metabolism; Oxidoreductases - genetics; Oxidoreductases - metabolism; Protein Binding
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M414018200

AMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also known as PRG3 (p53-responsive gene 3)) is a human caspase-independent pro-apoptotic protein with some similarity to apoptosis-inducing factor. AMID was purified from a recombinant bacterial host, enabling biochemical analysis of the protein. AMID is a flavoprotein; possesses NAD(P)H oxidase activity; and catalyzes NAD(P)H-dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. NADPH binds ∼10-fold tighter than NADH. AMID binds 6-hydroxy-FAD (a cofactor that accumulates only adventitiously and at low abundance in other flavoprotein enzymes) to form a stoichiometric cofactor·protein complex. AMID has a distinctive electronic spectrum due to the modified flavin. NAD(P)+ binding perturbed the spectrum, enabling determination of Kd values for these coenzymes. 6-Hydroxy-FAD could be removed from AMID and the apoprotein reconstituted with FAD. FAD was converted to 6-hydroxy-FAD in reconstituted AMID during aerobic turnover with NADPH. AMID is a DNA-binding protein that lacks apparent DNA sequence specificity. Formation of the protein·DNA complex (i) effected a major protein conformational change and (ii) was prevented in the presence of nicotinamide coenzyme. Apo-AMID retains DNA binding activity. Our studies establish a link between coenzyme and DNA binding that likely impacts on the physiological role of AMID in cellular apoptosis.