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 noncovalently. 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 detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins / E.M. Torchetti, F. Bonomi, M. Galluccio, E. Gianazza, T.A. Giancaspero, S. Iametti, C. Indiveri, M. Barile. - In: THE FEBS JOURNAL. - ISSN 1742-464X. - 278:22(2011), pp. 4435-4449. [10.1111/j.1742-4658.2011.08368.x]

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

F. Bonomi
Secondo
;
E. Gianazza;S. Iametti;
2011

Abstract

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 noncovalently. 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 detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.
conformational changes; FAD synthase; flavin binding; FMN adenylyltransferase; protein stability
Settore BIO/10 - Biochimica
2011
Article (author)
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/164698
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