Article

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

Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello (DBBM), Università degli Studi di Bari, Bari, Italy.
FEBS Journal (impact factor: 3.79). 09/2011; 278(22):4434-49. DOI:10.1111/j.1742-4658.2011.08368.x pp.4434-49
Source: PubMed

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.

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Keywords

'as isolated' protein
 
25 mixture
 
apo/holoprotein
 
client apoproteins
 
environmental conditions
 
enzyme binds 1 mole
 
enzyme catalyzes FAD assembly
 
FAD product
 
FAD release
 
FAD synthesis
 
hFADS2
 
human FAD synthase
 
isoform 2
 
lower rate
 
mild chaotropes
 
protein stability
 
rate-limiting step
 
recombinant His-tagged protein
 
structural rearrangements
 
whole catalytic cycle