(Received for publication, July 26, 1995; and in revised form, October 6, 1995)
From the
The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPH or NADH. Overexpression in E. coli now allows the preparation of large amounts of pure protein. Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain. Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein. On the other hand, the presence of the 2`-OH of the ribityl chain stimulates the NADPH-dependent reaction significantly. Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin. Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E. coli growing cells. As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.
Flavins are well known as key prosthetic groups of a large number of redox enzymes named flavoproteins. More recently, protein-free flavins, riboflavin, FMN, or FAD, were also suggested to play, as electron transfer mediators, important biological functions, for example during ferric iron reduction(1, 2, 3) , activation of ribonucleotide reductase(4, 5) , bioluminescence(6, 7) , and oxygen activation (8) (Fig. S1).
Figure S1: Scheme 1Biological functions of free flavins.
The reduction of free flavins by reduced pyridine nucleotides NADPH or NADH is not an efficient reaction. The kinetics is slow unless very high nonphysiological concentrations of both reactants are present in the reaction mixture(8) . As a consequence, living organisms have evolved enzymes that catalyze the reduction of riboflavin, FMN, and FAD by NADPH and NADH and are called NAD(P)H:flavin oxidoreductases or flavin reductases. It is now well established that such enzymes are present in all microorganisms, including the luminous marine bacteria, and also in mammals(1) . A recent study has shown that flavin reductase activities are present in erythrocytes and in various human tissues (liver, heart, kidney, and lung)(9) .
In most cases, a single living organism contains multiple flavin reductases different in enzymatic nature and molecular mass. The luminous bacteria, Vibrio harveyi, contains at least three types of FMN reductases(10, 11, 12, 13, 14) . In Escherichia coli at least two flavin reductases have been isolated. One, named Fre, is a 26.2-kDa enzyme using both NADH and NADPH as electron donors(4) , whereas the other is the sulfite reductase, a 780-kDa enzyme using NADPH exclusively(15) .
Still very little is known on the structure and the mechanisms of flavin reductases. No three-dimensional structure of such an enzyme is available yet, and only recently were the corresponding genes cloned, sequenced and overexpressed(9, 10, 11, 12, 16, 17, 18, 19) . Only in the cases of E. coli and V. harveyi were the enzymes obtained in a pure form and characterized(4, 13, 15) . In spite of such a limited information, it is nevertheless possible to propose a classification of flavin reductases in two groups.
In the first group, enzymes are flavoproteins, using a flavin prosthetic group for electron transfer from NAD(P)H to the flavin substrate. The prototype of this group is the NADPH-specific flavin reductase from V. harveyi (flavin reductase P)(13) . It is a monomer of 26 kDa with a tightly bound FMN cofactor and has been recently crystallized. Preliminary x-ray diffraction analysis of the protein has been reported(20) . The sulfite reductase of E. coli carries both FAD and FMN as cofactors and thus belongs to this group. This class of enzymes is characterized by ping-pong bisubstrate biproduct reaction mechanisms(14, 21) .
In the second group, enzymes do not contain any prosthetic group. The visible spectrum of the protein gives no evidence for a chromophore and excludes the presence of flavins. When added, FMN or FAD does not bind tightly, and the enzyme thus should not be classified as a flavoprotein. The prototype of this group is Fre, the NAD(P)H:flavin oxidoreductase from E. coli(4) . It consists of a single polypeptide chain of 233 amino acids, with a molecular mass of 26,212 Da. The NADH-specific FMN reductase enzyme from V. harveyi (flavin reductase D) belongs to this group and actually has significant amino acid sequence homology with Fre (48% identity) as well as a similar molecular weight(10) . A number of ferric reductases, for example from Rhodopseudomonas sphaeroïdes, Pseudomonas aeruginosa, or Neisseria gonorrhoeae, which absolutely require free flavins for activity, probably also belong to this group(22, 23) .
Such flavin reductases are interesting systems to study in that they utilize flavin only as a substrate and not as a coenzyme. It is thus important to understand how the polypeptide chain accomodates both reduced pyridine nucleotide and flavin to allow the electron transfer to proceed efficiently. It is also important to appreciate the structural basis for the difference in flavin recognition between flavoproteins and flavin reductases in order to delineate the different possible ways of interaction between the flavin and the polypeptide chain in relation with the function.
In this paper we have used a variety of flavin analogs (substrates and inhibitors) to probe the structural requirements of the flavin binding site of Fre, the NAD(P)H:flavin oxidoreductase from E. coli. Detailed kinetic studies of flavin reduction, in the presence of NADPH, show that the reductase follows a sequential mechanism, in agreement with the absence of a protein-bound mediator. Orders of substrate binding and product release have been determined. It is clearly established that the binding of the flavin occurs mainly through the isoalloxazine ring. In addition, lumichrome was found to be a very good inhibitor of the flavin reductase and to potentiate hydroxyurea as an inhibitor of ribonucleotide reductase and E. coli cell growth.
For 3,4-dimethylaniline derivatives 11a, b, and c, a mixture of 3,4-dimethylaniline (9.1 g; 75 mmol),
triethylamine (15 cm), and bromoalcohol (25 mmol) was
stirred at 110 °C for 5 h. After cooling and addition of
dichloromethane (2
100 cm
), the solution was washed
with an aqueous Na
CO
solution (10%; 40
cm
). The aqueous layer was extracted with dichloromethane
(200 cm
). The combined organic extracts were dried over
MgSO
and evaporated under reduced pressure. Compound 11a was purified by chromatography on silica gel eluting with a
dichloromethane/methanol mixture (98:2) (yield, 70% oil).
H
NMR (80 MHz, CDCl
)
7.02 (1H, d, J = 8
Hz, ArH); 6.53 (1H, s, ArH); 6.42 (1H, d, J = 8 Hz, ArH); 3.75 (2H, t,
CH
-OH or CH
-NH); 3.55 (2H, br
s, NH and OH); 3.27 (2H, t,
CH
-NH or CH
-OH); 2.25 (6H, s,
2 CH
).
C NMR (20 MHz,
CDCl
)
146.5; 137.0; 130.1 (ArCH); 125.5;
115.3 (ArCH); 110.8 (ArCH); 60.6 (CH
-OH or CH
-NH); 46.3 (CH
-NH or CH
-OH); 19.7 (CH
); 18.4 (CH
). MS (EI) m/e 165 (88, M
); 164 (100,
(M-1)
); 105 (50,
(M-NHCH
CH
OH)
). Compound 11b was obtained in a pure form after chromatography on silica gel
eluting with a dichloromethane/methanol mixture (98:2) or after
distillation under reduced pressure (yield, 80% oil). b.p.
107-110 °C (p = 0.1 mm Hg).
H NMR
(300 MHz, CDCl
)
6.98 (1H, d, J = 8
Hz, ArH); 6.70 (1H, s, ArH); 6.67 (1H, d, J = 8 Hz, ArH); 3.82 (2H, m,
CH
-OH or CH
-NH); 3.60 (1H, m,
NH or OH); 3.40 (1H, br s, OH or
NH); 3.32 (2H, m, CH
-NH or
CH
-OH); 2.19 (3H, s, CH
);
2.16 (3H, s, CH
); 1.90 (2H, m,
CH
).
C NMR (75 MHz, CDCl
)
146.3; 136.9; 130.0 (ArCH); 125.4; 115.0
(ArCH); 110.6 (ArCH); 61.0 (CH
-OH or CH
-NH); 42.1 (CH
-NH or CH
-OH); 31.8 (CH
); 19.8 (CH
); 18.4 (CH
). Compound 11c was purified by washing
of the crude residue with dichloromethane (yield, 64%). m.p.
100-102 °C.
H NMR (200 MHz,
Me
SO-d
)
6.79 (1H, d, J = 8 Hz, ArH); 6.38 (1H, d, J < 2 Hz,
ArH); 6.30 (1H, dd, J = 8 Hz, J <
2 Hz, ArH); 4.90 (1H, t, OH or NH); 4.68
(1H, d, J = 4.7 Hz, CHOH); 4.52 (1H, t, J = 5.6 Hz, NH or OH); 3.60 (1H, br s,
CHOH); 3.33 (2H, m, CH
-OH or
CH
-NH); 2.90 (2H, m, CH
-NH or
CH
-OH); 2.08 (3H, s, CH
);
2.04 (3H, s, CH
).
C NMR (50 MHz,
Me
SO-d
)
147.1; 136.1; 130.0
(ArCH); 123.0; 114.0 (ArCH); 109.7 (ArCH);
70.1 (CH-OH); 64.1 (CH
-OH or CH
-NH); 46.8 (CH
-NH or CH
-OH); 19.8 (CH
); 18.4 (CH
). MS (FAB [+], glycerol) m/e 196 (100, (M+1)
); 121 (14,
(M+1-CH
CHOHCH
OH)
).
For
6-(N-substituted anilino)uracil derivatives 12a, b, and c, compound 11 (21 mmol) was dissolved in
water (11c, 30 cm) or in a 1:1 water/dioxane mixture (11a, 35 cm
; 11b, 40 cm
). The
solution was heated at reflux under argon and stirred during addition
of 6-chlorouracil (1.03 g; 7 mmol). After 15 h of reflux and cooling,
the pH was increased to 11 by addition of aqueous NaOH (10%). For 12a and b, the resulting solution was extracted with
dichloromethane (3
100 cm
) to remove unreacted
starting compound 11. Aqueous HCl was added to the aqueous layer
to reach pH 3. The resulting precipitate was collected by filtration,
washed with water, and then crystallized from water. A second fraction
of 12a or 12b was obtained after evaporation of the
filtrates. The residue was stirred with methanol and filtered. Methanol
was evaporated, and the residual solid was crystallized from water
(yields: 12a, 50%; 12b, 87%). 12a: m.p.
250-251 °C.
H NMR (200 MHz,
Me
SO-d
)
10.35 (1H, br s,
NH); 10.0 (1H, br s, NH); 7.20 (1H, d, J = 8 Hz, ArH); 7.07 (1H, d, ArH); 7.00
(1H, dd, J = 8 Hz, ArH); 5.40 (1H, br s,
OH); 4.03 (1H, s, 5-CH); 3.70 (2H, m,
3`-CH
or 1`-CH
); 3.50 (2H, m,
1`-CH
or 3`-CH
); 2.22 (6H, s,
2 CH
).
C NMR (50 MHz,
Me
SO-d
)
163.6 (C=O);
155.0 (C=O); 151.0 (C
); 140.3; 137.9;
135.7; 130.6 (ArCH); 128.5 (ArCH); 124.9
(ArCH); 77.4 (5-CH); 58.7 (3`-CH
or 1`-CH
); 53.8 (1`-CH
or
3`-CH
); 19.3 (CH
); 18.9 (CH
). MS (EI) m/e 275 (63,
M
); 274 (83, (M-1)
). 12b: m.p. 238-240 °C.
H NMR (300 MHz,
Me
SO-d
)
10.30 (1H, br s,
NH); 10.00 (1H, br s, NH); 7.21 (1H, d, J = 8 Hz, ArH); 7.04 (1H, d, J < 2 Hz,
ArH); 7.00 (1H, dd, J = 8 Hz, J <
2 Hz, ArH); 4.80 (1H, br s, OH); 4.13 (1H, s,
5-CH); 3.65 (2H, t, 3`-CH
or
1`-CH
); 3.44 (2H, t, 1`-CH
or
3`-CH
); 2.49 (3H, s, CH
);
2.22 (3H, s, CH
); 1.60 (2H, m,
2`-CH
).
C NMR (50 MHz,
Me
SO-d
)
163.8 (C=O);
154.8 (C=O); 151.2 (C
); 139.7; 138.1;
135.8; 130.7 (ArCH); 128.5 (ArCH); 124.8
(ArCH); 77.2 (5-CH); 57.3 (3`-CH
or 1`-CH
); 48.1 (1`-CH
or
3`-CH
); 29.9 (2`-CH
); 19.4 (CH
); 19.0 (CH
). MS (FAB
[+], glycerol) m/e 290 (16,
(M+1)
); 232 (2,
(M+1-C
H
OH)
). 12c:
After the addition of aqueous NaOH (10%), the unreacted starting
compound 11c was removed by filtration and washed with water.
Aqueous HCl was added to the filtrate to reach pH 3. Compound 12c was collected by filtration and purified by crystallization from
water (yield, 65%). m.p. 237-238 °C.
H NMR (200
MHz, Me
SO-d
)
10.35 (1H, br s,
NH); 10.20 (1H, br s, NH); 7.20 (1H, d, J = 8 Hz, ArH); 7.06 (1H, d, J < 2 Hz,
ArH); 7.04 (1H, dd, J = 8 Hz, J <
2 Hz, ArH); 5.80 (1H, br s, 2`-CHOH); 4.87 (1H, t,
3`-CH
OH); 3.95 (1H, s, 5-CH); 3.65 (3H,
m, 1`-CH
and 2`-CH); 3.31 (2H, m,
3`-CH
); 2.50 (3H, s, CH
);
2.22 (3H, s, CH
).
C NMR (50 MHz,
Me
SO-d
)
163.7 (C=O);
155.7 (C=O); 151.0 (C
); 140.8; 138.1;
135.8; 130.7 (ArCH); 128.5 (ArCH); 124.9
(ArCH); 77.6 (5-CH); 69.7 (2`-CHOH); 62.9
(3`-CH
or 1`-CH
); 55.1
(1`-CH
or 3`-CH
); 19.4 (CH
); 19.0 (CH
). MS (FAB
[+], glycerol) m/e 306 (100,
(M+1)
); 231 (2,
(M+1-CH
CHOHCH
OH)
).
For
isoalloxazine 5-oxides 13a, b, and c, sodium
nitrite (1.7 g; 25 mmol) was added to a solution of compound 12 (5 mmol) in acetic acid in the dark. The mixture was stirred at
room temperature for 3 h, and then water (6 cm) was added.
The suspension was stirred again for 3 h, and the solvents were
evaporated under reduced pressure. 13a: The residue was washed
with water and then with methanol to yield the compound (yield, 76%).
m.p. 280-282 °C.
H NMR (200 MHz,
Me
SO-d
)
11.00 (1H, s,
NH); 8.07 (H, s, ArH); 7.87 (H, s, ArH);
4.87 (1H, m, CH
OH); 4.63 (2H, t,
2`-CH
or 1`-CH
); 3.76 (2H, m,
1`-CH
or 2`-CH
); 2.38 (3H, s,
CH
); 2.34 (3H, s, CH
). MS
(DCI, NH
+ isobutane) m/e 303 (30,
M
); 287 (100, (M-16)
). 13b:
The residue was washed with water and then crystallized from ethanol (2
liters) (yield, 75%). m.p. 260 °C (dec.).
H NMR (300
MHz, Me
SO-d
)
11.00 (1H, br s,
NH); 8.08 (1H, s, ArH); 7.79 (1H, s, ArH);
4.70 (1H, br s, CH
OH); 3.58 (2H, t,
3`-CH
or 1`-CH
); 3.55 (2H, m,
1`-CH
or 3`-CH
); 2.45 (3H, s,
CH
); 2.38 (3H, s, CH
); 1.89
(2H, m, 2`-CH
). MS (FAB[+], NBA) m/e 317 (90, (M+1)
); 301 (41,
(M+1-16)
). UV (H
O, 50 mM Tris-HCl, pH 7.5),
(
): 460 nm(5280). 13c: The residue was chromatographed on C18 reversed phase
eluting with water. A crystallization from ethyl acetate yielded pure
compound (yield, 82%). m.p. 239 °C (dec.). MS (FAB
[+], NBA) (
)m/e 333 (100,
(M+1)
), 259 (48,
(M-CH
CHOHCH
OH)
).
For
*isoalloxazines 3, 4, and 5, an aqueous solution
of dithiothreitol (1.4 g; 20 cm) was added to a suspension
of N-oxide 13 (2 mmol) in ethanol (13a and 13b, 500 cm
; 13c, 600 cm
). The
mixture was heated at reflux with stirring for 20 min under argon. The
solvent was evaporated under reduced pressure, and the residue was
crystallized from a water/ethanol mixture (50:50) for 3 and 4 (respective yields: 70% and 92%) and from ethyl acetate for 5 (yield, 76%). 3: m.p. 275-277 °C.
H
NMR (200 MHz, Me
SO-d
)
11.30 (1H,
br s, NH); 7.67 (2H, s, 2 ArH); 4.92 (1H, t,
CH
OH); 4.66 (2H, t, 2`-CH
or
1`-CH
); 3.80 (2H, m, 1`-CH
or
2`-CH
); 2.39 (6H, s, 2 CH
).
C NMR (75 MHz, Me
SO-d
)
160.8; 158.7; 156.9; 149.5; 147.2; 137.1; 134.0; 132.3; 131.0
(ArCH); 117.5 (ArCH); 57.3 (2`-CH
or 1`-CH
); 46.5 (1`-CH
or 2`-CH
); 21.6 (CH
);
20.5 (CH
). MS (DCI, NH
+
isobutane) m/e 287 (100, M
); 243
(45, (M-CH
CH
OH)
). 4:
m.p. 295 °C (dec.).
H NMR (200 MHz,
Me
SO-d
)
11.30 (1H, br s,
NH); 7.90 (1H, s, ArH); 7.78 (1H, s, ArH);
4.75 (1H, m, CH
OH); 4.61 (2H, t,
3`-CH
or 1`-CH
); 3.55 (2H, m,
1`-CH
or 3`-CH
); 2.39 (6H, s,
2 CH
); 1.89 (2H, m, 2`-CH
).
C NMR (75 MHz, Me
SO-d
)
159.8; 155.5; 150.1; 146.4; 139.9; 135.7; 133.8; 131.0
(ArCH); 130.7; 115.9 (ArCH); 57.3
(3`-CH
or 1`-CH
); 46.5
(1`-CH
or 3`-CH
); 29.7
(2`-CH
); 20.6 (CH
); 18.7 (CH
). MS (FAB [+], NBA) m/e 301 (84, (M+1)
); 243 (35,
(M-(CH
)
OH)
). UV
(H
O, 50 mM Tris-HCl, pH 7.5),
(
): 444 nm(12200).
5: m.p. 300 °C (dec.). H NMR (200 MHz,
Me
SO-d
)
11.29 (1H, s,
NH); 8.02 (1H, s, ArH); 7.87 (1H, s, ArH);
4.94 (2H, m, 2 OH); 4.72 (2H, t, 3`-CH
or
1`-CH
); 3.55 (2H, br s, 1`-CH
or 3`-CH
); 2.41 (6H, s, 2
CH
); 2.30 (1H, br s,
2`-CH).
C NMR (50 MHz,
Me
SO-d
)
161.0; 157.0; 151.1;
148.1; 137.5; 136.7; 134.9; 132.7 (ArCH); 131.5; 117.9
(ArCH); 69.2 (CH
); 64.1 (CH
); 48.8 (CH
); 21.6 (CH
); 19.5 (CH
). MS (FAB
[+], NBA) m/e 317 (65,
(M+1)
).
Figure 3:
A, lumichrome as a competitive inhibitor
for riboflavin. The enzyme activity was assayed as a function of
riboflavin concentrations using 180 µM NADPH in the
absence () or in the presence of 0.5 (
), 1 (
), or 2
µM (
) lumichrome. B, lumichrome as an
uncompetitive inhibitor for NADPH. The enzyme activity was assayed as a
function of NADPH concentrations using 15 µM riboflavin in
the absence (
) or in the presence of 1 (
), 3 (
), or 5
µM (
) lumichrome.
E. coli K12 was transformed by plasmid pFN3, which contains the fre gene inserted at the polylinker site of pJF119EH, and overexpression was tested in Luria-Bertani medium in the presence of IPTG. Overexpression was maximal when the cells were in the late log phase. The flavin reductase specific activity of E. coli K12 soluble extracts was around 50 units/mg of protein. Typically, extracts from E. coli K12 carrying pFN3 gave values of 5,000-7,500 units/mg after IPTG induction, thus showing a 100-150-fold overexpression of the enzyme. On the basis of a specific activity of 130,000 units/mg for the pure flavin reductase, it can be estimated that the overexpressed Fre enzyme represents 5% of the total soluble proteins. Extracts from E. coli K12 (pFN3) obtained in the absence of IPTG gave activity values of 300 units/mg, confirming the good control of pJF119EH derivative plasmids by the inducer. In addition, during growth, no significant loss of pFN3 in the presence or absence of IPTG has been noticed.
A two-step purification protocol, with a phenyl-Sepharose chromatography followed by gel filtration on Superdex 75, has been developed. From 4.5-liter cultures of E. coli K12 (pFN3) and 1-g protein extracts, 15-20 mg of about 90-95% pure flavin reductase, as judged by SDS-polyacrylamide gel electrophoresis, was obtained. The yield of the purification was 45%. The specific activity of the purified flavin reductase was 120,000 units/mg.
Figure 1:
A, flavin reductase activity as a function
of NADPH concentration in the presence of 1 (), 2 (
), or 4
µM (
) riboflavin. B, flavin reductase
activity as a function of riboflavin concentration in the presence of
24 (
), 36 (
), or 60 µM (
) NADPH. The
results are presented as double reciprocal plots with straight lines
determined by a linear regression program.
When the enzyme activity was
determined as a function of NADPH concentration in the absence or in
the presence of three concentrations of AMP, a dead-end inhibitor,
double reciprocal plots of values obtained reflected typical
competitive inhibition kinetics (Fig. 2A). A rather
large K value (0.5 mM) for AMP can be
determined from these lines. On the other hand, patterns of
noncompetitive inhibition was observed with respect to riboflavin (Fig. 2B).
Figure 2:
A, AMP as a competitive inhibitor for
NADPH. The enzyme activity was assayed as a function of NADPH
concentrations using 15 µM riboflavin in the absence
() or in the presence of 200 (
), 400 (
), or 600
µM (
) AMP. B, AMP as a noncompetitive
inhibitor for riboflavin. The enzyme activity was assayed as a function
of riboflavin concentrations using 180 µM NADPH in the
absence (
) or in the presence of 600 µM (
) or 2
mM (
) AMP.
Furthermore, lumichrome is both a strong
competitive inhibitor of riboflavin with a K value
of 0.5 µM (Fig. 3A) and an uncompetitive
inhibitor of NADPH (Fig. 3B). All these data support
the conclusion that the flavin reductase has an ordered mechanism with
NADPH binding first(28) .
The kinetic mechanism of product
release has been determined by studying inhibition by
products(29) . When NADPH concentration was varied with a fixed
concentration of riboflavin, inhibition by NADP was
found to be competitive with respect to NADPH with a K
value of 5 mM. When riboflavin was varied at a fixed
NADPH concentration, NADP
appeared to inhibit
noncompetitively (data not shown). This now suggests that the first
product to be released is the reduced flavin, followed by
NADP
.
Figure S2:
Scheme 2Structure of the riboflavin
derivatives and synthesis intermediates. Compounds 11, 12, and 13 were
prepared with different R chains referred to as a for R
= CH
-CH
OH, b for R
=
(CH
)
-CH
OH, and c for
R
= CH
-CHOH-CH
OH. Compound
13d was with R
=
1`-deoxyribityl.
On the
other hand, the theory shows that it is impossible to determine the K values for the flavin precisely. One has to rely
on K
values to analyze how changes in flavin
structure affects the flavin-protein interaction. In a first
approximation, we may consider that large variations in K
values roughly reflect variations in flavin
recognition by the enzyme when the substrates give k
values of the same order of magnitude.
As previously
reported(4) , the K values for riboflavin
and FMN were in the 1-3 µM range with either NADPH
or NADH. Riboflavin in both cases gave the highest k
value, but the differences between flavins were much greater with
NADPH. The presence of a terminal phosphate group on the ribityl chain,
as in FMN, greatly altered the catalytic efficiency of the reaction.
FAD gave no reaction with NADPH. Also the introduction of a
H-phosphonate group led to a large decrease of the k
value (compare compounds 4 and 6).
Lumiflavin,
with a methyl group at N-10, was also a substrate with K values similar to those of natural substrates and k
larger than that of FMN. This now shows that
the ribityl chain is not essential for recognition by the enzyme.
Accordingly, ribitol has no inhibitory effect on the NADPH-riboflavin
reductase activity even at 100 mM concentration. Moreover, as
shown above, lumichrome, which lacks the ribityl chain, is a very
efficient competitive inhibitor with respect to riboflavin.
On the
other hand, k but not K
can
be modulated significantly by chemical modification of the sugar chain.
Charge, discussed above, is not the only parameter, because a large
difference in k
is also observed between
riboflavin and lumiflavin. In order to get deeper insight into such a
modulation, we also tested a series of flavin derivatives in which the
sugar moiety has been modified. Table 1shows that the 2`-OH may
play a role in the NADPH-dependent reaction because similar catalytic
efficiency (k
/K
) is found
for riboflavin and compounds 3 and 5, whereas the
reaction was 2- and 5-fold less catalytically efficient with compound 4 and lumiflavin, respectively.
Because binding of the flavin molecule seems to occur mainly through the isoalloxazine ring, it is important to determine which sites of that ring are participating to the recognition of the molecule by the polypeptide chain.
First,
methyl groups at C-7 and C-8 seem to play an important role in the
binding because compound 8, the lumiflavin derivative that lacks
these methyls, is also a substrate but with a K value 10-fold larger than that for lumiflavin. Furthermore,
alloxazine, the lumichrome analog lacking the methyl groups, is also an
inhibitor but with a K
value about 200-fold larger
than that for lumichrome. The only compound in this study containing a
modification at N-5 was riboflavin N-oxide (13d). The
catalytic activity of the enzyme was not affected by the presence of
the oxo group (data not shown).
Methylation at the N-3 of riboflavin
(compound 7) greatly decreased the catalytic efficiency of the
reaction, due to both a large increase of the K value and a large decrease of the k
value.
Because N-3 plays only a limited role in flavin redox chemistry, with
redox potentials insensitive to N-3 alkylation, it appears that this
site may be involved in flavin binding(31) .
Finally, compounds 12, which contain both the dimethylphenyl and pyrimidine moieties, were totally devoid of inhibitory properties (data not reported), showing that the binding site of the enzyme has a specific requirement for the whole isoalloxazine ring.
Figure 4:
Inhibition of cell growth by lumichrome
and hydroxyurea. Growth of E. coli K12 or LS1312 under
standard conditions () or in the presence of 140 µM lumichrome (
), 40 mM hydroxyurea (
), or 40
mM hydroxyurea and 140 µM lumichrome
(
).
When E. coli K12 cells
were grown aerobically in M9 medium, the addition of 40 mM hydroxyurea at an OD of about 0.05 resulted, as
expected, in a significant decrease of the growth rate. Lumichrome,
instead, had no effect. When now a combination of 40 mM
hydroxyurea and 140 µM lumichrome was added to the culture
medium, bacteria stopped growing totally, indicating a strong and
remarkable synergic effect of the combination.
Similar behavior was observed when the flavin reductase was inactivated genetically. Growth of an E. coli mutant strain, LS1312, lacking an active fre gene was fully inhibited by addition of 40 mM hydroxyurea alone, confirming the function of the flavin reductase during repair of hydroxyurea-treated E. coli cells. As far as growth inhibition by hydroxyurea is concerned, addition of lumichrome or inactivation of the fre gene gave similar phenotypes.
In order to study the structure and the mechanism of the NAD(P)H:flavin oxidoreductase from E. coli, an overproducing strain of E. coli was obtained. The transformation of E. coli K12 by plasmid pFN3, which contains the fre gene under the control of the tac promotor, led, after IPTG induction, to a 100-150-fold overexpression of the enzyme. Such an overexpression allowed purification of the soluble enzyme by two chromatographic steps, phenyl-Sepharose and Superdex 75. Crystallization of such preparations is presently under investigation.
The NAD(P)H:flavin oxidoreductase from E. coli is the
prototype of the class of flavin reductases, which do not contain any
light-absorbing (flavins, metals) prosthetic group for mediating the
electron transfer from reduced pyridine nucleotide to free oxidized
flavin. The kinetic analysis of the enzymatic reaction demonstrated
that the flavin reductase has an ordered mechanism (Fig. S3).
The catalysis by the 26-kDa polypeptide chain is thus achieved by
providing a site where both substrates bind and interact. NADPH binds
first to the active site, followed by the flavin. After electron
transfer, the first product to be released is the reduced flavin,
followed by NADP. This is in full agreement with AMP
and NADP
being competitive inhibitors with respect to
NADPH and noncompetitive with respect to flavin and lumichrome being a
competitive inhibitor with respect to flavin and noncompetitive with
respect to NADPH. Similar results were obtained in the case of flavin
reductase D, the NADH-dependent flavin reductase from V. harveyi(32) .
Figure S3: Scheme 3Proposed reaction mechanism of the flavin reductase Fre.
The remarkable efficiency of lumichrome as an
inhibitor of the flavin reductase in vitro (K = 500 nM) led us to test whether it may also
affect the activity under in vivo conditions. As a matter of
fact, lumichrome greatly potentiated the inhibitory effect of
hydroxyurea, a specific inhibitor of ribonucleotide reductase, on the
growth of E. coli K12 cells(33) . This is a very
interesting observation because (i) it is in good agreement with the
observation that in bacteria, flavin reductase plays a crucial role in
the activation of hydroxyurea-inactivated ribonucleotide reductase and
(ii) it shows for the first time that the combination of an inhibitor
of ribonucleotide reductase such as hydroxyurea and an inhibitor of
flavin reductase might have potential applications for inhibition of
DNA synthesis and for general antiproliferative activity. Whether such
a strategy could be applied not only to microorganisms but also to
human beings, for example for cancer treatment, is of course just
speculative. It is important also to note that very recently
hydroxyurea has received renewed attention in the context of AIDS
research because it was found to greatly potentiate the anti-HIV
effects of nucleoside analogs such as
2`,3`-dideoxyinosine(34) .
The riboflavin substrate is
composed of two distinct regions, a highly hydrophobic isoalloxazine
ring and a ribityl side chain linked at N-10. The results reported in
this study show that the binding to the polypeptide chain occurs mainly
through the isoalloxazine ring as a whole. This is clear from the
following observations: (i) lumichrome, an analog of isoalloxazine with
no sugar chain, is a very good inhibitor with a low K (0.5 µM), indicating a strong binding to the enzyme,
while ribitol has no inhibitory properties; (ii) the rigidity of the
ring seems to be an important parameter, because compounds 12 have no inhibitory effects; (iii) similar K
values were found for riboflavin, lumiflavin, and for flavin
analogs with various side chains. The same K
values were found for FMN, FAD, and compound 6 in spite of
the presence of a negatively charged group and of the important size of
the side chain. In addition, the methyl groups at position 7 and 8
(compare the K
for lumichrome and alloxazine and
the K
values for lumiflavin and compound 8)
and nitrogen at position 3 (compare the K
values
for riboflavin and compound 7) play an important role in the
binding of the flavin ring.
In the absence of a three-dimensional
structure for the enzyme and for a substrate-enzyme complex, it is just
possible to use our data as a basis for predicting some structural
characteristics of the flavin reductase involved in flavin recognition.
The following analysis was based on the refined structures of
flavoproteins. In flavodoxin (35) and
ferredoxin-NADP reductase(36) , possessing FMN
and FAD, respectively, as an integral component of the protein,
aromatic residues (tryptophan or tyrosine) are involved in the
recognition of the isoalloxazine ring through
-orbital overlaps.
This seems to be a general strategy that may operate in the case of
flavin reductase as well. On the other hand, in flavoproteins,
extensive interactions also exist between the protein and the ribityl
phosphate or ribityl phosphate-AMP and contribute to the overall
affinity of the apoprotein for the cofactor significantly. These
interactions seem to be very weak in the case of the flavin reductase,
in agreement with the low specificity of the enzyme as well as its weak
affinity for flavin substrates.
On the other hand, as shown in Table 1, some of the hydroxyl groups of the ribityl chain seem to
slightly contribute to decreasing the activation barrier for catalysis.
Actually, in the presence of NADPH, the k value
for lumiflavin is about 5-fold lower than that for riboflavin. The
magnitude of the effect of the ribityl group is most conveniently
expressed as a decrease in the free energy of activation, calculated
from the corresponding k
values according to
G =
-RTln[k
(ribo)/k
(lumi)],
where k
(ribo) and k
(lumi)
refer to riboflavin and to lumiflavin. The decrease in the free energy
of catalysis is about 0.9 kcal/mol, indicating a limited but
nevertheless significant role of the sugar chain in enzyme catalysis.
From the substrates tested here, it is possible to identify the
important hydroxyl groups for this effect. The 4`- and 5`-OH do not
contribute at all because compound 5 had the same k
value as riboflavin. On the other hand, 2`-OH
and to a lesser extent 3`-OH may be involved in decreasing the
activation barrier of the reaction (compounds 3 and 4).
Catalysis seems to be also controlled by the charge of the side
chain. Actually with NADPH, which contains a pyrophosphate and a
phosphate group, as a reducing agent, the k value for FMN, which contains one phosphate group, is 20-fold
smaller than that for the neutral riboflavin, and moreover the enzyme
is unable to catalyze the reduction of FAD, which contains a
pyrophosphate group. Furthermore, weaker discrimination is obtained
with NADH (riboflavin:FMN:FAD = 1:0.37:0.37), which has one
phosphate group less than NADPH. The negative effect of a charge on the
flavin substrate is also seen from the low activity of compound 6.
In addition, it is interesting to note that, as an FMN reductase, Fre is more specific for NADH. This further supports the similarity to flavin reductase D, one of the flavin reductases from V. harveyi, which has been described, with FMN as the electron acceptor, to be specific for NADH(13) .