F. Fieschi et al., CYS5 AND CYS214 OF NAD(P)H-FLAVIN OXIDOREDUCTASE FROM ESCHERICHIA-COLI ARE LOCATED IN THE ACTIVE-SITE, European journal of biochemistry, 237(3), 1996, pp. 870-875
The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) fr
om Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes th
e reduction of free flavins by NADPH or NADH. A sequential ordered mec
hanism, with NADPH binding first, operates. Fre is the prototype of a
class of flavin reductases able to transfer electrons with no prosthet
ic group. It has been previously reported that several members of this
family, including Fre, were inactivated by thiol reagents such as N-e
thylmaleimide (MalNEt). Amino acid sequence similarities among these e
nzymes reveal that one of the three cysteines residues of Fre is highl
y conserved. Altogether this suggested a crucial role of cysteine resi
dues for catalysis. The three cysteine residues were mutated to serine
residues. Single-mutant and double-mutant enzymes were as active as t
he wild-type and K-m values for both substrates remained the same. Cys
teine residues are thus not important for activity. Nevertheless, we s
howed that cysteines 5 and 214, but not cysteine 149, were responsible
for MalNEt inactivation. In addition, it has been found that riboflav
in, but not NADPH, can protect Fre from MalNEt inactivation. This stro
ngly 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 N
ADPH.