Fieschi F, Nivière V, Fontecave M
Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, UMR C5616, CNRS, Université Joseph Fourier, Grenoble, France.
Eur J Biochem. 1996 May 1;237(3):870-5. doi: 10.1111/j.1432-1033.1996.0870p.x.
The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) from Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes the reduction of free flavins by NADPH or NADH. A sequential ordered mechanism, with NADPH binding first, operates. Fre is the prototype of a class of flavin reductases able to transfer electrons with no prosthetic group. It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N-ethylmaleimide (MalNEt). Amino acid sequence similarities among these enzymes reveal that one of the three cysteines residues of Fre is highly conserved. Altogether this suggested a crucial role of cysteine residues for catalysis. The three cysteine residues were mutated to serine residues. Single-mutant and double-mutant enzymes were as active as the wild-type and Km values for both substrates remained the same. Cysteine residues are thus not important for activity. Nevertheless, we showed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation. In addition, it has been found that riboflavin, but not NADPH, can protect Fre from MalNEt inactivation. This strongly suggested that Cys5 and Cys214 are located at the flavin-binding site of Fre and that flavin can bind to the enzyme in the absence of NADPH.
来自大肠杆菌的NAD(P)H:黄素氧化还原酶（NADPH:核黄素氧化还原酶）Fre是一种26.1 kDa的单体，它催化NADPH或NADH对游离黄素的还原反应。其作用机制为顺序有序机制，首先是NADPH结合。Fre是一类能够在没有辅基的情况下转移电子的黄素还原酶的原型。此前有报道称，该家族的几个成员，包括Fre，会被硫醇试剂如N-乙基马来酰亚胺（MalNEt）灭活。这些酶之间的氨基酸序列相似性表明，Fre的三个半胱氨酸残基之一高度保守。这表明半胱氨酸残基在催化过程中起着关键作用。将这三个半胱氨酸残基突变为丝氨酸残基。单突变和双突变酶的活性与野生型相同，两种底物的Km值也保持不变。因此，半胱氨酸残基对活性并不重要。然而，我们发现半胱氨酸5和214，而非半胱氨酸149，是导致MalNEt灭活的原因。此外，还发现核黄素而非NADPH可以保护Fre不被MalNEt灭活。这强烈表明Cys5和Cys214位于Fre的黄素结合位点，并且在没有NADPH的情况下黄素可以与该酶结合。
