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Báo cáo khoa hoc : Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins

A soluble form of human FAD synthase (isoform 2; hFADS2) was pro-duced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although non-covalently. Complete release of FAD from the ‘as isolated’ protein requires extensive denaturation. | IFEBS Journal Human FAD synthase isoform 2 a component of the machinery that delivers FAD to apo-flavoproteins Enza M. Torchetti1 Francesco Bonomi2 Michele Galluccio3 Elisabetta Gianazza4 Teresa A. Giancaspero1 Stefania Iametti2 Cesare Indiveri3 and Maria Barile1 5 1 Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello DBBM Universita degli Studi di Bari Bari Italy 2 Dipartimento di Scienze Molecolari Agroalimentari DISMA University degli Studi di Milano Milan Italy 3 Dipartimento di Biologia Cellulare Universita della Calabria Arcavacata di Rende Italy 4 Gruppo di Studio per la Proteomica e la Struttura di Proteine Dipartimento di Scienze Farmacologiche DSF Universita degli Studi di Milano Milan Italy 5 Istituto di Biomembrane e Bioenergetica IBBE CNR Bari Italy Keywords conformationalchanges FAD synthase flavin binding FMN adenylyltransferase protein stability Correspondence M. Barile Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello DBBM Universita degli Studi di Bari via Orabona 4 I-70126 Bari Italy Fax 39 080 5443317 Tel 39 080 5443604 E-mail m.barile@biologia.uniba.it Received 9 May 2011 revised 3 August 2011 accepted 23 September 2011 doi 10.1111 j.1742-4658.2011.08368.x A soluble form of human FAD synthase isoform 2 hFADS2 was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly although non-covalently. Complete release of FAD from the as isolated protein requires extensive denaturation. A 75 25 mixture of apo holoprotein could be prepared by treatment with mild chaotropes allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions the enzyme catalyzes FAD assembly from ATP and FMN and at a much lower rate the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in .