(Received for publication, April 20, 1995; and in revised form, July 16, 1995)
From the
Monoamine oxidase B (MAO B) catalyzes the oxidative deamination
of biogenic and xenobiotic amines. The oxidative step is coupled to the
reduction of an obligatory cofactor, FAD, which is covalently linked to
the enzyme at Cys. In this study, we developed a novel
riboflavin-depleted (Rib
) COS-7 cell line to study
the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO
B cDNA in these cells. We found that MAO B is expressed equally in the
presence or absence of FAD and that apoMAO B can be inserted into the
outer mitochondrial membrane. Flavinylation of MAO B was achieved by
introducing MAO B cDNA and different flavin derivatives simultaneously
into Rib
COS-7 cells via electroporation. Since the
addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B
activity, we conclude that the flavin which initially binds to apoMAO B
is FAD. In our previous work, we used site-directed mutagenesis to show
that Glu
in the dinucleotide-binding motif of MAO B is
essential for MAO B activity, and we postulated that this residue is
involved in FAD binding. In this study, we tested the role of residue
34 in flavin binding by expressing wild-type or mutant MAO B cDNA in
Rib
COS-7 cells with the addition of
[
C]FAD. We found that Glu
is
essential for both FAD binding and catalytic activity. Thus, FAD binds
to MAO B in a dual manner at Glu
noncovalently and
Cys
covalently. We conclude that Glu
is
critical for the initial non-covalent binding of FAD and is
instrumental in delivering FAD to the covalent attachment site at
Cys
.
The major amine-degrading enzymes in the central nervous system
and peripheral tissues of mammals are monoamine oxidase A and B (MAO ()A and B, amine:oxygen, oxidoreductase (deaminating,
flavin-containing), EC 1.4.3.4). These isozymes are integral proteins
of the outer mitochondrial membrane (1) and can be
distinguished by differences in substrate preference(2) ,
inhibitory specificity(3) , tissue and cell
distribution(4, 5, 6) , and immunological
properties(7, 8, 9) . Furthermore, comparison
of their nucleotide and deduced amino acid sequences show that human
MAO A and B are two distinct proteins encoded by different
genes(10) .
Oxidation of amines by MAO is coupled to the
reduction of an obligatory cofactor, FAD, which is covalently linked to
the enzyme. Five types of bonds are generally found in the covalent
linkage of flavins to their respective apoproteins(11) . These
include a histidine residue which can be attached through its N-1 or
N-3 atom to the 8-methyl group of the isoalloxazine ring to form a
tertiary amine; a cysteine residue which forms a thioether linkage with
either the 8
-methyl group or the C-6 of the xylene ring of the
flavin molecule; or a tyrosine residue can become linked to the
8
-methyl group to form an (O)-8
-flavin bond. In MAO
A and B, the 8
-methyl group of FAD is bound covalently to cysteine
through a thioether linkage in the pentapeptide
SGGCY(12, 13) . Comparison of this segment with the
complete deduced amino acid sequences of MAO A and B indicated that FAD
is covalently bound to Cys
in MAO A and Cys
in MAO B, respectively(10) . In addition, site-directed
mutagenesis studies of MAO B, where Cys
was substituted
with serine or histidine, showed that this cysteine residue is
essential for catalytic activity(14, 15) .
Although
the amino acid sequences surrounding the FAD covalent attachment site
in different flavoproteins bear little homology, a distinct
non-covalent FAD-binding site displays high sequence identity in many
FAD-containing enzymes of diverse function(16, 17) .
This non-covalent FAD binding region is commonly referred to as the
dinucleotide-binding site or motif due to its interaction with the AMP
moiety of FAD. This motif consists of a
-sheet-
-helix-
-sheet beginning
with a highly conserved Gly-X-Gly-X-X-Gly sequence
between the first
-sheet and the
-helix. The second
-sheet usually ends with a glutamate residue in which the
-carboxylate group is thought to interact through a hydrogen bond
with the 2`-hydroxyl group of ribose in the AMP moiety of FAD. In MAO A
and B, this motif is located at the N terminus of MAO A (residues
15-43) and MAO B (residues 6-34) and ends in Glu
and Glu
, respectively. Site-directed mutagenesis
studies, where Glu
was replaced with aspartate, glutamine,
or alanine, resulted in near complete or total loss of catalytic
activity in MAO B(18) .
A fundamental process in the
intracellular generation of functional flavoenzymes is the molecular
mechanism which generates holoenzyme from apoenzyme and its cofactor.
Following the discovery of the first known enzyme with covalently
linked FAD (succinate dehydrogenase, 19), extensive research in many
laboratories has been conducted to elucidate how FAD is coupled to its
respective proteins. The precise steps involved remain unknown. In this
study, we developed a novel riboflavin-depleted (Rib)
COS-7 cell line to investigate the flavinylation of MAO B. ApoMAO B was
obtained by expressing MAO B cDNA in these cells. We show that the
expression of MAO B apoenzyme is independent of FAD and that apoMAO B
can be inserted into the outer mitochondrial membrane. Coupling of
flavin to the apoenzyme was studied using FAD, flavin derivatives, or
[
C]FAD. We also examined the role of a critical
glutamate residue (Glu
) in flavinylation of MAO B using
site-directed mutants. We find that Glu
plays an essential
role in flavin coupling to the apoenzyme. We propose that the
dinucleotide-binding site at the N terminus of MAO B provides a
topological dock for the inital binding of FAD, and then FAD is
delivered to the covalent attachment site at Cys
.
Figure 1:
A, elution chromatogram of standards.
Pure ATP, FAD, FMN, and riboflavin (Sigma) were eluted on a C-18
Semi-Prep column with a linear gradient from 100% A/0% B (A = 10
mM (NH)
HPO
, pH 6.8; B
= acetonitrile) to 60% A/40% B in 20 min at a flow rate of 4
ml/min. The FAD standard was eluted at a retention time of 10 min. B, elution chromatogram of [
C]FAD. The
[
C]FAD peak eluted at a retention time of 10 min
using the same elution profile as above. C, elution
chromatogram of 8
-hydroxyriboflavin. Synthetic
8
-hydroxyriboflavin was isolated from the reaction mixture (see
``Materials and Methods'') and rerun on HPLC using the same
elution conditions as above. 8
-Hydroxyriboflavin gave a single
sharp peak shown on the chromatogram. The authenticity of
8
-hydroxyriboflavin was confirmed by spectroscopic analysis (UV
and mass spectrometry).
Synthesis of 8-hydroxyriboflavin was carried out by the
method of McCormick(21) . Synthetic 8
-hydroxyriboflavin
was resolved on HPLC to yield a major sharp peak on the chromatogram (Fig. 1C). The authenticity of 8
-hydroxyriboflavin
was further confirmed by spectroscopic analysis (UV and mass
spectrometry).
Figure 2:
The effect of riboflavin depletion in
COS-7 cells on MAO B enzymatic activity and MAO B expression. MAO B
cDNA was transfected into COS-7 cells at different time intervals
during the process of riboflavin depletion from the cells. The
expression level () and the MAO B activity (
) from these
cells were determined. The activity is expressed as the percentage of
the enzymatic activity of MAO B holoenzyme obtained in Rib
COS-7 cells.
Figure 3:
Western blot analysis. MAO B cDNA was
transfected in Rib COS-7 cells with the addition of
different cofactors via electroporation. Expressed MAO B enzymes were
adjusted to equal concentrations using ELISA before
immunoprecipitation. Immunoprecipitated enzymes were separated on 10%
SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by
Western blotting using the MAO B-specific monoclonal antibody MAO
B-1C2. Lane 1, prestained molecular mass marker; lane
2, MAO B obtained from transfected Rib
COS-7
cells, which served as a positive control. Lanes 3-8 contain MAO B obtained from transfected Rib
COS-7 cells with or without the addition of different cofactors. Lane 3, riboflavin; lane 4, FMN; lane 5,
FAD; lane 6, 8
-hydroxyriboflavin; lane 7,
NAD
; lane 8, no cofactor addition; lane
9, untransfected Rib
COS-7 cells; lane
10, biotinylated molecular mass
marker.
Figure 4:
In vitro flavinylation assays
(see ``Materials and Methods''). Triton extracted MAO B
holoenzyme from a transfected Rib COS-7 cell lysate,
which served as a positive control, remained fully active during 3 h
incubation at 30 °C (
). However, no MAO B catalytic activity
was observed when FAD was added after apoMAO B had been synthesized.
Triton extracted (
) or nonextracted (
) MAO B apoenzyme
from transfected Rib
COS-7 cell lysate were incubated
at 30 °C with exogenous FAD. Triton-extracted MAO B apoenzyme from
transfected Rib
COS-7 cell lysate were also incubated
at 30 °C with exogenous FAD, an energy mixture and with or without
25% glycerol (
, with glycerol;
, without glycerol). The
enzymatic activity of each sample was determined at 1-h time intervals
using [
C]benzylamine as
substrate.
Figure 5:
Fluorography. Wild-type and mutant MAO B
cDNAs were transfected in Rib COS-7 cells with the
addition of exogenous [
C]FAD. Expressed
wild-type and variant MAO Bs were adjusted to equal concentrations
using ELISA before immunoprecipitation. The immunoprecipitated enzymes
were separated on 10% SDS-PAGE and analyzed on fluorography. Lane
1,
C-methylated molecular mass marker; lane
2, wild-type MAO B; lane 3, E34A MAO B; lane 4,
E34Q MAO B; lane 5, E34D MAO B; lane 6, V10I MAO B; lane 7, untransfected Rib
COS-7 cells; lane 8,
C-methylated molecular mass
marker.
Flavinylation of MAO B has been difficult to study in the
past because FAD is covalently attached to Cys, and this
cofactor cannot be removed without sacrificing MAO B
activity(28) . For mammalian flavoproteins, the conventional
approach has been to study flavinylation in animals. Rabbits or mice
were fed riboflavin-free diets to deplete the endogenous riboflavin,
and the animals were sacrificed to obtain the organs or tissues for
analysis(29) . This method is time-consuming, tedious, and
subject to variation due to individual differences in animals. We have
now developed a convenient and rapid method to study flavinylation of
eucaryotic proteins in Rib
COS-7 cells. Since COS-7
cells are not capable of synthesizing riboflavin, enzymes expressed in
these cells lack flavin cofactors. Rib
COS-7 cells
were used to produce apoMAO B to study the steps involved in
flavinylation. In other related studies, Nishikimi et al.(30) produced the apoenzyme of L-gulono-
-lactone oxidase in a baculovirus expression
system in which riboflavin levels were reduced. Enzymatic activity was
observed upon addition of FAD, but no covalently bound FAD could be
obtained using this system.
The expression level (0.95 ± 0.04
µg/mg protein) of MAO B in transfected RibCOS-7
cells remained unchanged in sequential transfections during the process
of riboflavin depletion (Fig. 2). This observation indicates
that MAO B expression is not dependent upon riboflavin or FAD
concentrations in the cell. The level of expressed MAO B was determined
by ELISA, which is based upon epitope recognition by antibodies and is
susceptible to major conformational changes. Both MAO B-1C2 monoclonal
antibody and goat anti-MAO B polyclonal antibodies were capable of
recognizing apoMAO B. In another study, the apoenzyme of bacterial
6-hydroxy-D-nicotine oxidase, which contains covalently bound
FAD in its holoenzyme, is not recognized by a molecular chaperone as
aberrant(31) . We suggest that the conformation of the apoMAO
B, like apo-6-hydroxy-D-nicotine oxidase, may be similar to
that of the native holoenzyme.
Mitoma and Ito (32) found
that the mitochondria targeting sequence of MAO B is located on the C
terminus of the molecule. Deletion of the C-terminal 28 amino acids of
MAO B abolished transfer of the enzyme to the mitochondria, while
deletion of the N-terminal 55 amino acids had no effect on
mitochondrial targeting. Furthermore, an expressed hybrid protein, in
which the C-terminal 29 amino acids of MAO B was fused to the
hydrophilic portion of cytochrome b, was localized
in the mitochondria. In our work, we found that apoMAO B expressed in
Rib
COS-7 cells was localized in the mitochondrial
fraction of cell lysates (Table 2), indicating that bound FAD is
not necessary for MAO B insertion into the mitochondria membrane. These
results are consistent with those of Mitoma and Ito (32) and
support the notion that the target C-terminal sequence alone is
sufficient for insertion into the membrane.
One advantage of using
Rib COS-7 cells to study flavinylation is that
exogenous FAD or its derivatives can be introduced with MAO B cDNA into
the cells during the transfection process. The enzymatic activity of
MAO B with the addition of different flavins can be determined and
compared with MAO B holoenzyme expressed in Rib
COS-7
cells (Table 3). Addition of FAD resulted in the restoration of
about 75% of holoMAO B activity. Interestingly, approximately 75% of
holoMAO B activity was also achieved by the addition of riboflavin or
FMN to transfected Rib
COS-7 cells, suggesting the
presence of abundant levels of cellular FAD synthetase. The addition of
8
-hydroxyriboflavin gave an enzyme with 40% activity of the
control, which raises the possibility that this flavin may represent an
intermediate in the activation of FAD (discussed below). Full recovery
of MAO B enzymatic activity obtained in Rib
COS-7
cells was not achieved for reasons that remain unknown. In related
studies, however, Brandsch and Bichler (33) found that the
covalent flavinylation of 6-hydroxy-D-nicotine oxidase in
vitro required specific effectors (phosphorylated three carbon
compounds), such as glycerol 3-phosphate, glyceraldehyde 3-phosphate,
or glycerate 3-phosphate. Effectors that could enhance the activity of
MAO B have not been identified. We speculate that the achievement of
only 75% of activity may be due to a slight change in metabolism of
Rib
COS-7 cells which have been adapted to grow in
riboflavin-free medium for more than 100 days.
Although it is known
that FAD is covalently attached to active MAO B molecules, the form of
the flavin which initially binds to MAO B in vivo has not
previously been established. Theoretically, riboflavin or FMN could
first bind to apoMAO B followed by phosphorylation and adenylation,
respectively, to form FAD. If riboflavin or FMN is the form that
initially binds to apoMAO B, we would expect FAD binding to be much
less effective than riboflavin or FMN. Since MAO B activity was
recovered to approximately the same extent (75%) using FAD, FMN, or
riboflavin, we conclude that the flavin moiety which initially binds to
apoMAO B is FAD. Apparently, FAD synthetase in these cells rapidly
converted riboflavin and FMN to FAD by phosphorylation and adenylation,
respectively, prior to incorporation. The presence of FAD was confirmed
by measuring the covalent binding of [C]FAD to
MAO B.
The covalent attachment of FAD to Cys could be
autocatalytic or catalyzed by an as yet uncharacterized enzyme. In
either case, one of the participants, the 8
-methyl group of the
flavin moiety or Cys
of MAO B, must be activated prior to
coupling. Although the nucleophilicity of the Cys
residue
may be influenced by surrounding amino acid residues, it is difficult
to envision that a cysteine derivative would react with the inert
8
-methyl group of the flavin moiety. From a chemical point of
view, activation of the 8
-methyl group appears essential for
coupling of FAD to apoMAO B. An enzymatically facilitated pathway for
the incorporation of FAD into flavoproteins has been proposed by Decker (11) in which a flavin cofactor may be enzymatically activated
by hydroxylation of the 8
-methyl group, followed by
(pyro)phosphorylation (Fig. 6). Since the (pyro)phosphate is a
good leaving group, a simple S
2 reaction could facilitate
the formation of the thioether between the flavin moiety and MAO B. To
test this hypothesis, we synthesized the putative activated
intermediate 8
-hydroxyriboflavin and determined in Rib
COS-7 cells its ability to generate MAO B enzymatic activity
(synthesis of 8
-phosphate-riboflavin was also attempted, but was
unsuccessful because the highly reactive hydroxyl groups on the ribityl
moiety were also phosphorylated). If the flavin derivative is truly an
intermediate, we assumed that it would be capable of entering the
flavinylation pathway to produce active MAO B. MAO B activity was
obtained, but the level was only about half of that obtained with the
addition of riboflavin (Table 3). One possible explanation for
the low activity is that a flavinylating enzyme binds the flavin
substrate and catalyzes hydroxylation and phosphorylation sequentially
without release of the 8
-hydroxy intermediate. Thus, the
8
-hydroxy intermediate may not be recognized as efficiently as
riboflavin during the initial binding step. Alternatively, the covalent
flavinylation of MAO B may be autocatalytic, since the unactivated form
of the flavins (riboflavin, FMN, and FAD) has higher efficiency of
incorporation into apoMAO B than the putative activated form. Studies
by Weyler et al.(34) support the concept that
flavinylation of MAO may be autocatalytic, based on the observation
that MAO expressed in yeast cells (Saccharomyces cerevisiae)
is active and contains covalently bound FAD. Since yeast cells do not
contain any known enzymes with covalently linked flavin, they reasoned
that the cells are unlikely to contain any flavinylating enzymes which
could have catalyzed the coupling reaction in MAO.
Figure 6:
A
hypothetical mechanism of covalent bond formation postulated by
Decker(11) . This mechanism involves the enzymatic activation
of the 8-methyl group of the isoalloxazine ring of a flavin
cofactor by hydroxylation and (pyro)phosphorylation, followed by
covalent attachment to the apoenzyme (MAO B in this
case).
Studies were also
conducted to determine whether flavinylation occurs as a
co-translational or post-translational process. When FAD and MAO B cDNA
were added simultaneously to Rib COS-7 cells, active
MAO B (containing FAD) was obtained. However, when FAD was added in
vitro to whole cell lysates after apoMAO B was synthesized, MAO B
activity could not be regenerated (Fig. 4). Furthermore, when
apoMAO B was extracted from the mitochondrial membrane, attempts to
regenerate active flavinylated MAO B were unsuccessful, even in the
presence of various energy mixtures and glycerol (Fig. 4). The
inability to couple FAD to apoMAO B in vitro may indicate that
flavinylation occurs as a cotranslational process during elongation of
nascent chains to form functionally competent MAO B molecules.
Our
previous work demonstrated that Glu in the dinucleotide
binding motif was critical for MAO B catalytic activity (18) .
Two variants at Glu
(E34A and E34Q) were devoid of
enzymatic activity, and another conservative variant, E34D, had only 7%
of the wild-type activity. It was not known, however, whether the role
of Glu
is confined to alignment of FAD for participation
in the oxidation-reduction cycle of catalysis, or is involved in FAD
incorporation. In this study, we show that the loss of activity in
Glu
variants is linked to the inability to bind FAD
covalently (Fig. 5).
Since FAD binds to two regions of MAO B
(noncovalently at Glu and covalently at
Cys
), the absence or low levels of FAD incorporation into
Glu
variants reveals an important feature of the
flavinylation process. If FAD coupling occurred by initial covalent
attachment to Cys
, Glu
variants would
contain covalently bound FAD, but would be inactive because FAD could
not interact properly at the dinucleotide-binding site. Since we find
little or no covalent binding of FAD in the Glu
variants,
we conclude that FAD binds to Glu
first. We propose that
the dinucleotide-binding site (including Glu
) provides a
topological dock for the initial binding of FAD and is instrumental in
the delivery of FAD to Cys
in MAO B. The incoming flavin
cofactor, which is initially bound to the dinucleotide-binding site of
MAO B, could be held for a finite time in a position which places the
8
-methyl group of FAD in exact and close proximity to Cys
to facilitate covalent flavinylation.
The dinucleotide-binding sites in various flavoproteins contain high sequence identity(17) . However, the location within the primary structure varies from protein to protein, indicating that this site performs an autonomous function of cofactor binding within a heterologous group of flavoproteins. Furthermore, in many flavoproteins containing dinucleotide-binding sites, FAD is not covalently bound (17) . Our finding that the dinucleotide-binding site in MAO B plays a role in initial FAD binding indicates that this site alone is sufficient for a flavoprotein to bind a flavin cofactor. The significance of covalent linkage between FAD and its flavoenzyme remains unresolved, but covalent binding could play a role in enzyme integrity and stability, substrate stereospecificity, cofactor economy, or redox potentials. Understanding the MAO flavinylation process may lead to the design of MAO enzymes with high redox potentials for better catalysis and to the rational design of MAO inhibitors. Since MAO inhibitors have long been used for the treatment of various psychiatric and neurological disorders, including depression (35) and Parkinson's disease(36) , our studies on flavinylation may lead to the development of therapeutic drugs (analogs of FAD) for these disorders.