van Berkel Laboratory of Biochemistry, Wageningen University, the Netherlands Flavoproteins are ubiquitous redox proteins that are involved in many biological processes.In the majority o
Trang 1R E V I E W A R T I C L E
Deflavination and reconstitution of flavoproteins
Tackling fold and function
Marco H Hefti*, Jacques Vervoort and Willem J H van Berkel
Laboratory of Biochemistry, Wageningen University, the Netherlands
Flavoproteins are ubiquitous redox proteins that are
involved in many biological processes.In the majority of
flavoproteins, the flavin cofactor is tightly but noncovalently
bound.Reversible dissociation of flavoproteins into
apo-protein and flavin prosthetic group yields valuable insights in
flavoprotein folding, function and mechanism.Replacement
of the natural cofactor with artificial flavins has proved to be
especially useful for the determination of the solvent
acces-sibility, polarity, reaction stereochemistry and dynamic
behaviour of flavoprotein active sites.In this review we
summarize the advances made in the field of flavoprotein
deflavination and reconstitution.Several sophisticated
chromatographic procedures to either deflavinate or
reconstitute the flavoprotein on a large scale are discussed.In
a subset of flavoproteins, the flavin cofactor is covalently attached to the polypeptide chain.Studies from riboflavin-deficient expression systems and site-directed mutagenesis suggest that the flavinylation reaction is a post-translational, rather than a cotranslational, process.These genetic approaches have also provided insight into the mechanism
of covalent flavinylation and the rationale for this atypical protein modification.
Keywords: apoprotein; deflavination; FAD; flavin; flavo-enzyme; flavoprotein; FMN; (metal) affinity chromato-graphy; reconstitution.
Introduction
Flavoproteins are ubiquitous proteins that use flavins as
prosthetic groups.The common flavin cofactors are FMN
and FAD, which are synthesized in vivo from riboflavin
(vitamin B1) by the action of riboflavin kinase [1,2] and
FAD synthetase [3].The redox active isoalloxazine moiety
of the flavin cofactor may undergo one- or two-electron
transitions [4].This property and the ability to catalyse a
wide range of biochemical reactions [5,6] make flavoproteins
to be at the crossroads of cellular redoxchemistry.During
the past 60 years, an impressive amount of flavoproteins has
been characterized and many details about their
cata-lytic and structural features have been determined.In this
period, Vincent Massey was the leading character in flavin
research [7].
In most flavoproteins, the flavin is tightly but
noncova-lently bound.However, in a subset of flavoproteins, the
flavin is covalently attached to the polypeptide chain [8].
Nature facilitated the binding of flavins to proteins by the evolution of different flavin binding folds [9–12].Within these folds, specific parts of the flavin molecule are recognized by different structural motifs, like for instance the TIM barrel and the Rossmann fold.Many proteins that
do bind FMN do not interact with FAD, and vice versa, and this is reflected in their fingerprint sequences [13–20] Some flavoproteins contain both a FMN- and a FAD-binding domain.Well-studied examples of these diflavin enzymes include NADPH-cytochrome P450 reductase [21], nitric oxide synthase [22,23], flavocytochrome P450 BM3 [24], methionine synthase reductase [25,26], sulfite reductase [27–31], glutamate synthase [32,33] and dihydropyrimidine dehydrogenase [34].
In this paper, we present an overview of the field of flavoprotein deflavination and reconstitution.This topic is
of central interest to flavin enzymology as it provides valuable insights in flavoprotein folding, function and mech-anism.Earlier reviews in this field have concentrated on the methods of apoflavoprotein preparation [35–37], the use of artificial flavins [38], and the functional role and mechanism
of covalent flavinylation [8,39].Here, new methods of flavoprotein reconstitution are described and combined with insights obtained from the structural and functional analysis of mutant enzymes.In the first part of this review, the thermodynamics of flavin binding and the value of flavin analogs are briefly discussed.Then, the old vs.new approaches of reversible flavin removal are evaluated Finally, attention is given to the reconstitution of proteins containing covalently bound flavins.A comprehensive overview of the kinetics and thermodynamics of flavo-protein reconstitution is beyond the scope of this article and only selected cases are discussed.
Correspondence toW.J.H.van Berkel, Laboratory of Biochemistry,
Wageningen University, Dreijenlaan 3, 6703 HA Wageningen,
the Netherlands.Fax: + 31 317 484801,
E-mail: willem.vanberkel@wur.nl
Abbreviations: DAO,D-amino-acid oxidase; HAP, hydroxyapatite
chromatography; VAO, vanillyl-alcohol oxidase; PHBH,
p-hydroxy-benzoate hydroxylase; Nbs2, 5,5¢-dithio-bis(2-nitrop-hydroxy-benzoate);
IMAC, immobilized metal-affinity chromatography
*Present address: Key Drug Prototyping BV, Wassenaarseweg 72,
2333 AL Leiden, the Netherlands
Dedication: dedicated to Vincent Massey (1926–2002)
(Received 17 June 2003, accepted 21 August 2003)
Trang 2Thermodynamics of flavin binding
The binding interaction between apoproteins and flavin
prosthetic groups has been studied extensively.The strong
and specific binding of FMN or FAD to apoflavoproteins is
driven by the enthalpic contribution to the free energy
change of binding [40–42].The thermodynamics of flavin
binding to flavodoxin have been studied in detail [42–46].
Carlson and Langerman [44] showed that the enthalpy,
entropy and free energy associated with the binding of
FAD, 8-carboxyriboflavin, or the natural cofactor FMN to
Azotobacter vinelandii apoflavodoxin were all negative.
However, striking differences in the enthalpy for binding
FMN, FAD and 8-carboxyriboflavin ( )28.3, )16.6, and
)14.0 kcalÆmol)1, respectively) were observed.It was
concluded that the difference in binding enthalpy between
FMN and 8-carboxyriboflavin results from the phosphate
group in FMN, which binds in a predefined binding pocket
in the apoflavodoxin at the N-terminus of an a-helix.The
unique hydrogen bonding network surrounding the
FMN-phosphate group stabilizes the FMN-apoprotein complex
[47].This observation was recently corroborated by Lostao
et al.[42], who dissected the binding energies of the
Anabaena apoflavodoxin-FMN complex.It was shown that
the contribution of the phosphate to the binding energy is
the greatest (7 kcalÆmol)1), that the contribution of the
isoalloxazine is around 5–6 kcalÆmol)1, and that the ribityl
side chain contributes only 1 kcalÆmol)1.For flavodoxin
from Desulfovibrio vulgaris it was found that riboflavin only
binds to the apoprotein in the presence of inorganic
phosphate (Fig.1) Moreover, co-operative effects were
observed linked to the binding of inorganic phosphate and
the 5¢-phosphate of FMN [48].It was proposed that
phosphate binding induces a conformational switch,
creating a population of apoflavodoxin that is capable of binding the isoalloxazine ring [49].
The thermodynamics of FAD binding toD-amino-acid oxidase (DAO) has been studied by Matteo and Sturtevant [40].The free energy of binding was shown to be largely independent of temperature.However, the enthalpy and the entropy of the binding interaction were strongly tempera-ture dependent.In contrast to the binding of FMN to apoflavodoxin, where the entropy strongly opposes bind-ing, the binding of FAD to DAO is enforced by a large positive entropic contribution.It was proposed that this is due to a decrease in the exposure of nonpolar groups to the solvent and a smaller negative entropic contribution resulting from a tightening of the protein structure with losses in (vibrational) heat capacity when the coenzyme is bound.In general it is clear that (preformed) hydrogen bonding networks are important in flavin-binding, resulting
in large negative enthalpy factors and that the entropic contribution to flavin binding can be either negative in rigid apoproteins (flavodoxins) or positive in more flexible apoproteins [40].
The enthalpic and entropic contributions of flavin binding are very important, when dealing with apoflavo-protein preparation.First the enthalpic contribution should
be decreased in order to release the flavin from the holo-protein and second the entropic contribution in the prepar-ation of the apoprotein should be decreased.The enthalpic contribution in flavin binding can be decreased by the addition of solutes that interfere with the specific inter-actions between flavin cofactor and apoprotein.These solutes can be unfolding agents (urea, guanidinium hydro-chloride) which break hydrogen bonds, or monovalent anions (chloride, bromide) which influence protein hydra-tion.Phosphate and pyrophosphate can be used addition-ally [50] as these compounds can bind specificaddition-ally in the phosphate or pyrophosphate binding pocket of FMN and FAD containing flavoproteins, thereby diminishing the binding interaction of the flavin prosthetic group with the apoprotein [45,51].Often a combined effect of urea and bromide provides the best result in the preparation of apoflavoproteins.In those cases where the apoprotein is relatively unstable, it is important to reduce the entropic contribution in the preparation of apoflavoproteins.Many flavoproteins are less stable when they loose their cofactor and care should be taken when removing the flavin prosthetic group.An attractive deflavination approach is
to bind the protein to a chromatographic support.This diminishes entropic contributions by increasing the rigidity
of the apoprotein.
Flavin analogs
In order to gain insight into how the protein environment influences the reactivity of the flavin, it is desirable to remove the native prosthetic group in a, for the protein, nondestructive way.The flavin prosthetic group can be replaced with an artificial [38,52–56] or isotopically enriched analog [57–69].Replacement with a flavin analog should result in the (functionally active) reconstituted holoprotein FMN and FAD analogs can be synthesized conveniently from riboflavin, either chemically [1] or enzymatically [3], and can be isotopically enriched [70].
Fig 1 Crystal structure of Desulfovibrio vulgaris flavodoxin The
protein is depicted in green, the riboflavin moiety of FMN in yellow
and the 5¢-phosphate moiety in lime green
Trang 3Artificial flavins have proved very useful in the
determin-ation of the solvent-accessibility, polarity, reaction
stereo-chemistry, and dynamic behaviour of flavoprotein active
sites [38].Artificial flavins can be used to study the relative
distance between particular flavin atoms and the protein,
especially when using a series of flavins which are modified
at a specific position, thus varying the Vanderwaals radius
of the substituent.The solvent accessibility of the
dimethyl-benzene and pyrimidine part of the isoalloxazine ring system
can be probed by the introduction of a reactive
(photo-active) group [71–76].Artificial flavins can also be used to
covalently link the flavin to the protein [77], or to determine
the nature of amino-acid residues in the flavin binding site
[52,72,78,79].The chemical modification of the flavin
molecule can have drastic influences on its reactivity,
spectral properties or redox potentials.This provides a
powerful tool to study the enzyme reaction mechanism and/
or the flavin–apoprotein interaction [38,80–82].
Reconstitution experiments with 8-mercaptoflavins are
useful to get information about the polarity of the
flavoprotein active site.Binding of 8-mercaptoflavins to
apoflavoproteins may result in strong absorption spectral
changes, indicative for the stabilization of either the
benzoquinoid or thiolate anion form [38].Crystal structures
of several flavoprotein oxidases have confirmed the original
conclusion [38] that in these enzymes, the benzoquinoid
resonance form of the 8-mercaptoflavin is stabilized by a
positive charge, localized near the N1–C2 region of the
isoalloxazine ring.
The absolute stereochemistry of flavoproteins can be
determined by replacing the natural cofactor with
5-deaza-5-carba-8-demethyl-8-hydroxyflavin [3,83].Incorporation
of this artificial flavin allows the chemical analysis of the
stereochemistry of hydride transfer which is, for kinetic
reasons, not possible with the natural flavin.The absolute
stereochemistry has been assigned in this way for a large
number of flavoenzyme reactions, with the crystal structure
of glutathione reductase serving as the standard [3,83].
Flavin analogs modified in the ribityl side chain may also
provide insight into flavoprotein function and mechanism
[54,84–87].For medium-chain acyl-CoA dehydrogenase,
it was shown that the replacement of natural FAD by
2¢-deoxy-FAD reduces the activity of the enzyme about a
millionfold [84].This strongly supported the view that the
2¢-hydroxyl group of the flavin ribityl chain is involved in
the stabilization of the partial negative charge of the
carbonyl oxygen of the acyl-CoA substrate in the transition
state [84,88,89].
Isotopically enriched flavins are suitable to get a detailed
view into the molecular and submolecular structure of the
protein-bound flavin molecule.13C and15N NMR chemical
shifts can reveal both p electron density, conformational
changes and dynamic behaviour of the flavin moiety, as well
as the presence of specific hydrogens at the carbon and
nitrogen atoms investigated.13C and 15N have a natural
abundance of 1.1% and 0.4%, respectively Therefore, the
flavoprotein has to be reconstituted with 13C- and 15
N-enriched flavins.This approach gives a detailed electronic
view of the single atoms of the isoalloxazine ring system of
the flavin prosthetic group in the various redox states and of
reaction intermediates formed [49,60,70,90,91].In the
flavo-proteins studied by NMR, it was observed that, as a rule,
the isoalloxazine ring carries a negative charge at the N(1) atom in the hydroquinone state.Moreover, in most flavoproteins the isoalloxazine ring is observed to be nearly planar in both the oxidized and the two-electron reduced state.A beautiful example of the value of NMR spectro-scopy in flavoprotein research was the identification of Intermediate II of the bacterial luciferase reaction [90].In this study the presence of C(4a)-hydroperoxyflavin could be established unambiguously, which was a major step forward
in understanding flavoprotein function.
Preparation and reconstitution of apoflavoproteins
There are many methods for the preparation of reconsti-tutable apoflavoproteins.Some already exist for over
60 years, other are more recently developed.They are all based upon either weakening of flavin binding or stabilizing the apoprotein formed.Established strategies for protein deflavination include: lowering the pH, increasing the salt concentration, changing the solvent and increasing the temperature.As the final goal is to obtain highly reconsti-tutable apoprotein in high yield, the approach should be chosen with care.Prolonged exposure to non-native condi-tions, for instance by dialysis, can lead to irreversible denaturation of the apoprotein.Therefore, the time needed for flavin removal should be relatively short.
As each (apo)flavoprotein has its own characteristics, several strategies for the reversible removal of flavins from flavoproteins have emerged.Initial deflavination protocols were based upon precipitation, partial unfolding, or dialysis
of the protein [35].More recent techniques focus on the binding of the protein to a chromatographic support, facilitating the removal of the flavin, and reconstitution of the apoprotein [36,92].
When studying the properties of apo or reconstituted flavoprotein, one needs to consider the side-effects of residual flavin in the endproduct.If replacement with a flavin analog is desired, residual natural flavin might influence the catalytic properties of the reconstituted enzyme considerably.The presence of residual flavin can even be more problematic when investigating the physical and spectroscopic properties of the apoprotein.
Below we describe several apoflavoprotein preparation procedures, starting with conventional methods.Then we turn to the growing field of immobilization-based deflavin-ation methods in which one uses specific characteristics of the holo flavoprotein to obtain the corresponding apopro-tein.As a guide, the methods of apoflavoprotein prepar-ation are listed in Table 1.
Conventional methods
In 1935, Theorell was the first who reported that flavopro-teins could be reversibly resolved into their constituents apoprotein and prosthetic group.To weaken the binding of the flavin, Old Yellow Enzyme was dialysed at pH 2.7, thus releasing the noncovalently bound FMN [93].A few years later, it was reported thatD-amino acid oxidase (DAO) [94] and the yeast enzyme of Haas [95] could release their flavin
in the presence of high concentrations of ammonium sulfate, a kosmotropic salt.Based on these initial findings
Trang 4a more common procedure for the preparation of
apo-flavoproteins was developed by combining a low pH with
a high ammonium sulfate concentration [96].The acid
ammonium sulfate precipitation method appeared to be
especially useful for recalcitrant flavoenzymes such as
lipoamide dehydrogenase from pig heart [97] and glucose
oxidase from Aspergillus niger [96] although with both
enzymes partial irreversible denaturation occurred, and
variable amounts of reconstitutable apoprotein were
obtained.Strittmatter showed that addition of a high
concentration of potassium bromide to the acid ammonium
sulfate solution weakens the electrostatic interactions
between the flavoprotein and the flavin.This increased the
apoprotein yield of cytochrome b5 reductase [98] and
oxynitrilase [99].Further addition of charcoal, to adsorb the
free flavin, resulted in an even more efficient removal of the
prosthetic group [55,100,101].
Treatment with trichloroacetic acid is another
precipita-tion method to resolve flavoproteins into apoflavoprotein
and prosthetic group.This easy to perform method was
developed for flavodoxin [102] and works also very well for
the related FMN-binding domain of cytochrome P450 BM3
[103].The precipitated apo forms of both these proteins can
withstand the extreme acidic conditions applied and dissolve
readily at neutral pH.
Many flavoproteins irreversibly aggregate at low pH.
Therefore, a procedure of apoprotein preparation was
developed based on dialysis against halide anions at
physio-logical pH.Flavoproteins which bind the flavin prosthetic
group rather weakly can be deflavinated using a high concentration of bromide ions [104–110].Chloride is less chaotropic and therefore less effective in removal of the flavin [111].Stronger chaotropes such as cyanide, cyanate and thiocyanate have been used as well, but with these nucleo-philic agents significant conformational perturbations pre-venting holoprotein reconstitution may occur [109,112,113] Addition of a phosphodiesterase or phosphatase to dilute solutions of holoflavoprotein shifts the equilibrium to the apo form, because free FAD or FMN is hydrolysed to FMN and riboflavin, respectively.These reactions are relatively fast, but not very useful for large scale apoprotein preparation [114].Moreover, care must be taken to remove the cleavage enzyme.
Ultrafiltration [115] and gel filtration [116] are more efficient than dialysis for large scale apoflavoprotein pre-paration.This is especially important when using extreme conditions.Apoglucose oxidase, for instance, can be prepared by acidification to pH 1.4–1.8, followed by gel filtration in the presence of 30% glycerol [55,62,117].Based
on far-UV circular dichroism data it was suggested that under these conditions, the apoprotein retains a compact fold with a high degree of native-like secondary structure [118].Nevertheless, reconstitution of apoglucose oxidase with FAD is incomplete and about 50% of irreversibly aggregated apoprotein is generally obtained [62,117,119] The reconstitution of apoglucose oxidase with FAD-analogs
is of substantial importance for the construction of enzyme-electrodes which can be used as biosensor devices [120].
Table 1 Procedures of apoflavoprotein preparation
Conventional methods Chromatographic methods
Acid ammonium sulfate precipitation Ion exchange chromatography
Glucose oxidase [96] Egg white riboflavin binding protein [140,141] Lipoamide dehydrogenase [97] Egg yolk riboflavin binding protein [142] Trichloroacetic acid precipitation Hydrophobic interaction chromatography Flavodoxin [102] Lipoamide dehydrogenase [128,151,153] FMN domain cytochrome P450 BM3 [103] Glutathione reductase [150]
With phosphodiesterase Butyryl-CoA dehydrogenase [150]
p-hydroxybenzoate hydroxylase [114] DNA photolyase [152]
With guanidinium hydrochloride L-Amino-acid oxidase [160]
Lipoamide dehydrogenase [121] Flavocytochrome b2 [60]
Cytochrome P450 BM3 [122] Hydroxyapatite chromatography
With urea p-Cresol methylhydroxylase [165]
Salicylate hydroxylase [123] Vanillyl-alcohol oxidase [166,167]
Lactate oxidase [124] Dye affinity chromatography
Cytochrome P450 reductase [21] Flavocytochrome b2 [172]
With halide ions D-Amino-acid oxidase [173]
Old yellow enzyme [93] p-Hydroxybenzoate hydroxylase [174]
D-Amino-acid oxidase [104] Covalent chromatography
Cytochrome b5 reductase [98] p-Hydroxybenzoate hydroxylase [114,239] Oxynitrilase [99] Immobilized metal affinity chromatography Xanthine oxidase [130] NifL PAS domain [69]
Gel filtration
Glucose oxidase [117]
Carbon monooxide dehydrogenase [137]
Hydroquinone hydroxylase [116]
Ultrafiltration
Cytochrome P450 reductase [115]
Trang 5Another conventional procedure of apoprotein
prepar-ation is to partially unfold the holoprotein with
guani-dinium hydrochloride [121,122] or urea [21,123,124].
A disadvantage of this method is that one needs to find
conditions in which the partially unfolded apoprotein is
capable of refolding.Circular dichroism spectroscopy
[46,118,125–127] and fluorescence spectroscopy [127–129]
are useful here to probe the folding behaviour of the protein
of interest.
For many metalloflavoproteins, most of the conventional
apoflavoprotein preparation procedures cause extensive
denaturation.A likely explanation for this is that
deflavin-ation and reconstitution of flavoproteins is difficult if the
quaternary structure is more complex, i.e when the protein
contains more cofactors and/or subunits.The
molybdo-iron-sulfur flavoprotein xanthine oxidase [130,131] can be
deflavinated by dialysis at physiological pH in the presence
of 2 M calcium chloride [132].The FAD released is
hydrolysed to FMN by the high concentration of salt
present.Other methods are based upon this technique, and
include calcium dicarbonate [133] or potassium iodide [134].
Carbon monoxide dehydrogenase from Oligotropha
carb-oxidovorans [135,136] is another molybdo-iron-sulfur
flavo-protein for which a specific procedure of flavin exchange
was developed [137].The flavoprotein subunits can be
removed from this a2b2c2 enzyme by dissociation with
sodium dodecyl sulfate.The recombinant flavoprotein
component of carbon monoxide dehydrogenase produced
in Escherichia coli is a fragile monomer that does not bind
FAD.However, when the monomeric apoflavoprotein is
complexed with the metalloprotein components, the
result-ing heterohexameric form of the enzyme readily integrates
FAD [137].This shows that the reconstitution of the native
enzyme involves structural changes that translate into the
conversion of the apoflavoprotein from non-FAD binding
to FAD binding.
Chromatographic procedures
For several flavoproteins, the conventional methods of
apoprotein preparation are satisfactory (Table 1).A good
example is the trichloroacetic acid precipitation procedure
developed for flavodoxins [102].Multidimensional NMR
studies have shown that the extreme conditions for
apoflavodoxin preparation do not lead to any structural
perturbation in the reconstituted holoprotein, compared to
the native protein [138,139].With many flavoproteins, and
especially when large amounts of apoprotein are required
(affinity-based) chromatographic procedures are the
meth-ods of choice.One of the advantages of these methmeth-ods is
that on-column protein aggregation is unlikely to occur, as
each molecule is at a relatively large distance from its
neighbours.Particularly during partial unfolding, this helps
stabilizing the apoform of the protein, before flavin
reconstitution.
Ion-exchange chromatography
Many flavoproteins can be reversibly adsorbed to an
ion-exchange support.However, for successful on-column
flavin removal, conditions are needed where the protein
still interacts with the ion-exchanger (low ionic strength) but
not with the flavin (low pH).This concept was first worked out for the riboflavin-binding proteins from chicken egg white [140,141] and chicken egg yolk [142,143].The holo forms of these carrier proteins can be separated in the apo forms and free riboflavin by cation-exchange chromatogra-phy at pH 3.7 [144] At this pH, riboflavin is released from the column whereas the apoprotein remains tightly bound The apoprotein can subsequently be released from the column by raising the pH and ionic strength of the elution buffer.
Unlike most other flavoproteins, aporiboflavin-binding protein interacts strongly with a large number of flavin derivatives but not with FMN or FAD [141].The structure
of chicken egg white riboflavin-binding protein is rather unusual [145].Besides from an N-terminal ligand-binding domain that is strongly conditioned by nine disulfide cross-links, it contains a flexible phosphorylated motif with nine phosphoserines, which is essential for vitamin uptake [146] The isoalloxazine ring of the tightly bound riboflavin molecule is stacked in between two tryptophans, explaining the strong fluorescence quenching observed upon flavin binding.
The interaction between aporiboflavin-binding protein and riboflavin in the oxidized and two-electron reduced state has been addressed by reconstitution of the protein with 13C- and 15N-enriched riboflavin derivatives [147] These studies revealed that the pKaof the N1 atom of the flavin in the reduced state is unusually high (pKa¼ 7.45) The effective binding of riboflavin to the apoform of riboflavin-binding protein has also received biotechnolo-gical attention.Aporiboflavin-binding protein was shown
to scavenge riboflavin in model beer solutions, thereby inhibiting the light-induced formation of reactive oxygen species and sunstruck off-flavour [148,149].
Hydrophobic interaction chromatography
To circumvent severe protein loss using conventional techniques, a generally applicable immobilization procedure was developed for the large scale preparation of apoflavo-proteins using hydrophobic interaction chromatography (HIC) [150].This method makes use of the fact that many flavoproteins bind to phenyl agarose at neutral pH in the presence of 1Mammonium sulfate.Entropy is the driving force in this process.After immobilization, the flavin can be removed by the addition of high concentrations of potas-sium bromide and/or lowering the pH of the elution buffer The HIC method has been successfully applied for a number of flavoproteins [150,151], sometimes with slight modifications, to get optimal results [37,60,152].
The HIC method for preparing apoflavoproteins works very well for prokaryotic and eukaryotic disulfide reduc-tases, including lipoamide dehydrogenase, glutathione reductase, and mercuric reductase, and is preferred over classical methods [150].For apolipoamide dehydrogenase it was shown that the kinetics of holoenzyme reconstitution are dependent on the source of enzyme [151] and on the type
of flavin [153].Initial FAD binding to the monomeric apoprotein results in dichlorophenol-indophenol activity and quenching of tryptophan fluorescence.Then, dimeriza-tion occurs as reflected by the lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of
Trang 6the visible absorption spectrum [151].For lipoamide
dehydrogenase from A vinelandii, the conformational
sta-bility of the monomeric apoprotein was compared with that
of the dimeric holoenzyme [128].Unfolding of the
apo-enzyme in guanidinium hydrochloride follows a simple
two-state mechanism and is fully reversible.However, the
midpoint of unfolding of the monomeric apoprotein (Cm
guanidinium hydrochloride ¼ 0.75M) is much lower than
that of the dimeric holoenzyme (Cmguanidinium
hydro-chloride ¼ 2.4M).Guanidinium hydrochloride unfolding
was also used to probe the conformational stability of
A vinelandii lipoamide dehydrogenase in the different redox
states [128].From this and additional mutagenesis studies it
was inferred that overreduction by NADH promotes
subunit dissociation and that the C-terminus of the protein
plays an important role in dimer stabilization [154–156].
Sometimes the apoprotein interacts very strongly with the
HIC column material and apoprotein elution or on-column
reconstitution can be difficult.In such cases, the apoprotein
may undergo irreversible conformational damage, e.g by
local unfolding or subunit dissociation.This was observed
to some extent with the apoprotein of butyryl-CoA
dehydrogenase from Megaspheara elsdenii [150], which
can not be reconstituted when bound to the column.By
using high concentrations of ethylene glycol as eluent, stable
apoprotein showing negligible residual activity could be
isolated with 50–80% yield.Spectral analysis of the
apoprotein revealed that the coenzyme A persulfide ligand
present in the native protein [157] is removed during
apoprotein preparation.At pH 7.0 and 25 C, the
apopro-tein is a mixture of dimers and tetramers, and reassociates to
a native-like tetrameric form in the presence of FAD.The
reconstitution with FAD is relatively slow, and is stimulated
in the presence of CoA ligands.Binding of CoA ligands
stimulates tetramerization of the reconstituted holoenzyme
and improves protein stability.This is in agreement with the
crystal structure of butyryl-CoA dehydrogenase [158] which
shows that the inhibitor acetoacetyl-CoA binds in an
extended conformation near the dimer–dimer interface.
Fluorescence/polarization experiments revealed that the
reconstituted protein is somewhat less stable than the native
holoprotein, and that FAD dissociates more easily [150].
Another protein that was successfully deflavinated by the
HIC method isL-amino-acid oxidase from the venom of
Crotalus adamanteus, the eastern diamondback rattlesnake
[159]. L-amino-acid oxidase is a dimeric glycoprotein,
containing one FAD per monomer, that catalyses the
oxidative deamination ofL-amino acids.The deflavination
method for L-amino-acid oxidase [160] is similar to the
original protocol developed for lipoamide dehydrogenase.
The apo form ofL-amino-acid oxidase remains bound to
the HIC column, while the FAD cofactor is washed away in
a buffer with 1.5Mammonium sulfate.The apoprotein of
L-amino-acid oxidase can be reconstituted with FAD
on-column.However, the FAD-reconstitutedL-amino-acid
oxidase is inactive, having a perturbed conformation of the
flavin binding site.In the presence of 50% glycerol the
reconstituted L-amino-acid oxidase becomes nearly
com-pletely active.It was suggested that repulsion of glycerol
from hydrophobic surfaces of the protein and the
simulta-neous interaction with protein polar regions initiates the
restoration of the internal protein hydrophobic core.Thus,
glycerol can have a restorative effect on the proposed partially unfolded equilibrium intermediates, and acts as a molecular chaperone [160].This function of glycerol was first demonstrated for the His30Leu mutant of pig kidney DAO [161,162].The replacement of His307 with leucine perturbs the active site conformation accompanied by weakening the protein–flavin interaction and decreasing the enzymatic activity.The negative effect of this mutation can be eliminated in the presence of glycerol, resulting in up
to 50% activity recovery and more than 16-fold increase of flavin affinity.From this it was concluded that glycerol assists in the rearrangement of the protein towards the holoprotein conformation, as well as in reducing the solvent accessibility of the protein hydrophobic core [163] Hydroxyapatite chromatography
Hydroxyapatite chromatography (HAP) is often used as a final step in protein purification.For preparation and reconstitution of apoflavoproteins, HAP has the advantage that high salt concentrations, which can stimulate apo-protein formation, are not necessarily a limitation.The interaction between the protein and hydroxyapatite is primarily the result of non-specific electrostatic interactions between the positively charged protein amino groups and the negatively charged column material [164].The deflavi-nation process is influenced by the charge distribution of the protein, as well as the kind of salt that is used as eluent.The HAP method of apoflavoprotein preparation was used for a recombinant form of the flavin-binding subunit of p-cresol methylhydroxylase from Pseudomonas putida [165] and for the His61Thr variant of vanillyl-alcohol oxidase (VAO) from Penicillium simplicissimum [166,167].Binding the His61Thr variant to hydroxyapatite appeared to be a very gentle and efficient method of obtaining the VAO apopro-tein.Upon washing with 200 mM phosphate buffer, the His61Thr protein remains tightly bound to the column, whereas the FAD is easily removed.This removal of FAD is superior to other methods.For instance, when the His61Thr holoenzyme is gel filtrated in the absence or presence of high salt, almost no flavin is released.Another advantage of the HAP method is that the His61Thr VAO apoprotein can be eluted in concentrated form by washing the column with
600 mMpotassium phosphate buffer.
While native VAO forms primarily octameric assemblies
of 507 kDa, the apoHis61Thr variant exists in solution as a dimer of 126 kDa.Binding of FAD or ADP to the dimeric apoenzyme induces octamerization.In contrast, incubation with riboflavin or FMN does not stimulate octamer assembly, suggesting that upon FAD binding, small conformational changes in the ADP-binding pocket of the dimeric VAO mutant are transmitted to the protein surface, thus promoting oligomerization [167].
Dye-affinity chromatography Polyaromatic dyes can bind to proteins that use a cofactor with a nucleotide moiety [168].Cibacron Blue has a strong affinity for the Rossmann bab dinucleotide binding fold [125,169], and many flavoproteins possess such a fold, either for binding the ADP part of FAD and/or NAD(P) [170] This feature can be used to separate apo and holo
Trang 7flavoproteins [171].Such a separation was performed with
Cibacron Blue Sepharose for flavocytochrome b2 [172], and
for DAO [173].
Red-A agarose, another polyaromatic dye-containing
material, has been used for the deflavination of
p-hydroxy-benzoate hydroxylase (PHBH) from Pseudomonas
fluores-cens [174].Under low ionic strength conditions, the red dye
of the column binds to the enzyme, displacing the FAD.The
column is then eluted with high-ionic strength buffer
containing the artificial flavin 6-azido-FAD, which binds
to the protein and displaces the dye The 6-azido-FAD
cofactor can be covalently linked to the protein by
irradiation.Enzyme that has not been photolabeled is
separated from the covalently photolabeled enzyme by
applying the reaction mixture to a Red-A column again.At
low ionic strength, the nonlabeled enzyme binds to the
column material, whereas the photolabeled enzyme passes
directly through the column [77,80].
Covalent chromatography
When partial unfolding of the holoprotein is required to
weaken the protein–flavin interaction, the above mentioned
chromatographic methods for protein deflavination may
not work properly because of the presence of high
concentrations of unfolding agents.Therefore, we
intro-duced the concept of covalent enzyme immobilization for
improving the yield and quality of the apoprotein of PHBH
from P fluorescens [114].
PHBH from P fluorescens is a homodimeric
FAD-dependent monooxygenase that contains 5 sulfhydryl groups
per monomer [175].Cys116 is the only solvent exposed thiol
group, accessible to N-ethylmaleimide and
5,5¢-dithio-bis(2-nitrobenzoate) (Nbs2) [176].Using this property, it is
possible to bind the enzyme covalently to a Nbs2–agarose
column [114].Oxidation [92] or mutation [177] of Cys116
does not influence catalysis but prohibits binding of the
protein to the Nbs2 column.In Fig.2, the PHBH dimer is
shown together with the solvent accessibility of the Cys116
sulfur atom.After coupling to the column material, the FAD
can be efficiently released from the protein with urea and KBr.The resulting apoprotein is eluted from the column after reaction with dithiothreitol.On-column reconstitution
of the apoprotein with an artificial or isotopically labelled flavin analog is possible as well.The high resolution crystal structure of PHBH reconstituted with arabino-FAD [54] has clearly shown that the covalent disulfide-exchange procedure
of apoprotein preparation is very mild and does not lead to any significant structural perturbation.
The thiol affinity chromatography method requires the presence of a freely available thiol group at the protein surface, making the method not generally applicable Although the introduction by mutagenesis of a surface accessible cysteine is in principle possible with every flavoprotein, this approach might sometimes be a long shot.Furthermore, the newly introduced cysteine should not interact intra or intermolecularly with other cysteines For preparation of apoPHBH, the original Nbs2-agarose has recently been replaced by commercially available thiopropyl sepharose [81,178,179].
Immobilized metal-affinity chromatography Recombinant DNA technology has opened the possibility
to add affinity tags to proteins, facilitating protein purifi-cation.In immobilized metal-affinity chromatography (IMAC), a polyhistidine tag at either the N-terminal or C-terminal end of the protein allows the strong binding of the protein to the column bound metal ion.This interaction
is only disrupted by high concentrations of imidazole, acidic
pH or chelating agents such as EDTA.A his-tagged flavoprotein bound to an IMAC column is in principle able
to withstand rather harsh conditions that can be used to successfully remove the flavin prosthetic group.
The IMAC method of apoflavoprotein preparation was developed for the flavin-containing PAS domain of NifL, a redox-sensing protein from A vinelandii [180,181].Deflavi-nation was achieved on a nickel-nitrilotriacetic acid column
by exploiting the available N-terminal His-tag [69].Protein-bound FAD was removed efficiently by washing the column
Fig 2 3D-structure of PHBH The FAD cofactor is shown in green.In the right panel, all amino-acid residues within a distance of 10 A˚ to the Cys116 sulfur atom (yellow) are shown.The solvent accessibility of the sulfur atom is indicated with yellow dots, and Cys116 is drawn with Vanderwaals-radii
Trang 8with KBr and urea.The apoprotein could be eluted from
the column with imidazole, but slowly precipitated after
column release.Therefore, on-column reconstitution was
performed by circulating a solution of 2,4a-13C2-FAD or
2,4a-13C2-FMN (Fig.3).
The reconstituted PAS domain containing a new flavin
cofactor was eluted from the column with imidazole.
Reconstitution of the flavoprotein is highly efficient, as
shown by NMR and UV-VIS absorption spectroscopy [69].
Due to the immobilization of the apoprotein during the
process of de- and reflavination, no severe protein loss is
observed.Although the NifL PAS domain normally binds
FAD, the His-tag reconstitution method unambiguously
showed that this protein also tightly interacts with FMN.
13C and31P NMR experiments confirmed that there is no
significant difference in the active site between the FAD- or
the FMN- reconstituted protein in either the reduced or
oxidized state [182].The use of IMAC for the preparation
and reconstitution of His-tagged apoflavoproteins might
become a general method as His-tag incorporation in
recombinant proteins is easily achieved.
Deflavination of covalent flavoproteins
Most flavoproteins bind their cofactor in a noncovalent
mode.However, in about 10% of the flavoproteins, the
isoalloxazine ring of the flavin is covalently linked to either a
histidine, tyrosine, or cysteine residue [8,39].Members of the
VAO family [18] have a remarkable tendency for covalent
anchoring of the flavin cofactor, whereas flavoproteins with
a Rossmann fold [10,183] prefer a noncovalent flavin
binding mode.Succinate dehydrogenase [184–186],
fuma-rate reductase [187,188], and monoamine oxidase [189–191]
are the classical examples of Rossmann fold enzymes that
contain a covalently bound flavin cofactor.
Several strategies have been used to get insight into the
mechanism of covalent flavinylation.One of these strategies,
developed for human monoamine oxidase, makes use of a
yeast strain auxotrophic for riboflavin and expression of the
enzyme in this strain in the presence of different riboflavin analogs [192,193].Another more generally applied strategy
is based on the replacement of crucial amino-acid residues
by site-directed mutagenesis [194–198].In short, these investigations have supported early proposals from model system studies [199,200] that covalent flavinylation involves
an autocatalytical iminoquinone-methide addition mechan-ism with flavin binding preceding covalent attachment [8,39].In line with this mechanism it was found that the apoenzyme of monoamine oxidase B, expressed in COS cells devoid of riboflavin, is correctly inserted into the outer mitochondrial membrane [201].
For succinate dehydrogenase from yeast it was esta-blished that flavinylation takes place within the mitochond-rial matrix after import of the flavoprotein subunit and the cleavage of a leader peptide [186,202,203].Moreover, flavinylation of this iron-sulfur flavoenzyme was enhanced
in the presence of the chaperone protein hsp60 [186] p-Cresol methylhydroxylase is an 8a-O-tyrosyl-FAD containing flavocytochrome involved in the anaerobic microbial degradation of alkylphenols.From individual expression of the heme- and flavin-binding subunits it was revealed that the apoflavoprotein component of p-cresol methylhydroxylase is capable of noncovalently binding FAD but that the interaction with the heme-containing subunit is required for the self-catalytic flavinylation reac-tion [204,205].
The rationale for covalent flavinylation is not always clear.It has been proposed that the covalent linkage is involved in (a) stabilization of the apoprotein structure (b) steric alignment of the cofactor in the active site to facilitate catalysis (c) modulation of the redox potential of the covalent flavin [39] and (d) suppression of unwanted side reactions [206].For fumarate reductase from E coli it was found that the replacement of His44 by either Arg, Cys, Ser
or Tyr results in correctly assembled protein variants that are fully saturated with noncovalently bound FAD [207,208].Based on the diminished activity with fumarate and the inability to oxidize succinate, it was suggested that the absence of the covalent linkage alters the redox potential
of the flavin.More recently, this proposal received experi-mental support from studies on other flavoproteins [209,210].For VAO from Penicillium simplicissimum it was shown that the covalent linkage between the C8a atom
of the isoalloxazine ring of the flavin and the N3 atom of His422 increases the redox potential of the flavin, thereby facilitating substrate oxidation [209].Furthermore, from crystallographic analysis of noncovalent VAO variants and the VAO apoprotein, it could be established that the flavin binds in this enzyme to a preorganized binding site and that His61 activates the neighbouring His422 for covalent binding of FAD [166,209] (see Fig.4).
A new type of flavin attachment was recently reported for the NqrB and NqrC subunits of the Na+-translocating NADH-quinone oxidoreductase from Vibrio alginolyticus [211,212].From MALDI-TOF MS analysis of proteolytic digests, it was concluded that in both NqrB and NqrC subunits, the FMN cofactor is attached by its 5¢-phosphate moiety to a threonine side chain.In agreement with this, no covalent flavin was detected in the Thr225Leu mutant of NqrC from Vibrio cholerae [213].From sequence compari-sons it was predicted that this novel type of phosphoester
Fig 3 Schematic representation of the preparation and reconstitution of
His-tagged apoflavoproteins by immobilization on a
nickel-nitrilotri-acetic acid column (from [69], with permission)
Trang 9binding between FMN and the apoprotein is conserved in
the NADH-quinone oxidoreductase sodium pumping
sys-tems of a number of marine and pathogenic bacteria and
that in some of these systems the target threonine is replaced
by a serine residue [214].
Conclusions and future perspectives
Since the pioneering work of Theorell [93], many methods
have been developed for the (large scale) preparation and
reconstitution of apoflavoproteins.Conventional
precipita-tion methods are rapid but the yield and reconstitutability of
apoprotein may vary dramatically.More recently developed
chromatographic procedures have the advantage that the
apoprotein is stabilized by immobilization, and that large
amounts of apo- or reconstituted flavoproteins can be
obtained.
His-tagged flavoproteins can be purified, deflavinated
and reconstituted on the same IMAC column.Therefore,
the use of IMAC for flavoprotein deflavination and
reconstitution should be further exploited.An interesting
possibility to facilitate flavin release with such a column is to
deflavinate the protein in its reduced state.In the reduced
form, flavin binding usually is less tight than in the oxidized
form [215,216].The introduction of a solvent exposed
cysteine residue is another engineering strategy that allows
the covalent binding of a flavoprotein to a solid support.
This method produces high quality apoflavoproteins
with-out affinity tags in very good yield [114].
Site-directed mutagenesis can be used to change the
strength of the flavin–apoprotein interaction.With such
approach, it is important that the amino-acid replacements
(or deletions) do not result in dramatic changes in protein
stability and/or enzyme catalysis.For instance, a surface
loop of 14 amino-acid residues was removed from
Rhodotorula gracilis DAO by rational design [217].This
shifted the protein from the dimeric to the monomeric
state.In the monomeric state, the FAD interacts more
weakly with the protein.The DAO mutant apoprotein was
then obtained by dialysis in the presence of a chaotropic agent [106].
Sometimes, site-directed mutagenesis is necessary to improve the flavin–apoprotein interaction.WithL-aspartate oxidase, a FAD-dependent enzyme involved in the micro-bial biosynthesis of NAD+, the protein could only be crystallised in the apoform [218].Attempts to crystallise the wild-type enzyme in the holo form were unsuccessful However, the Arg386Leu mutant, in which an active site arginine is replaced, turned out to be amenable to crystal-lization and structure elucidation in the active FAD-bound state [219].
Understanding the specific interaction between flavopro-teins and their cofactors is also of medical relevance.Since the earlier finding that a reduced affinity of human glutathione reductase for FAD due to a mutation can lead
to nonspherocytic haemolytic anaemia [220], several genetic defects affecting flavin binding have been described.Muta-tions causing impaired flavin binding have been reported for, e.g NADPH-oxidase [221–225], NADH:cytochrome b5 reductase [226–228], methylenetetrahydrofolate reductase [229–234], and dihydropyrimidine dehydrogenase [235] and the consequences at a molecular level are starting to emerge Apoptosis-inducing factor is a flavoprotein that can stimu-late a caspase-independent cell-death pathway required for early embryonic morphogenesis [236,237].To gain further insight into the redox properties of apoptosis-inducing factor, Lys176 and Glu313, located near the isoalloxazine ring of FAD, were individually changed into alanine by site-directed mutagenesis.Both apoptosis-inducing factor variants appeared to be highly active when assayed in the presence of excess FAD.However, during purification the Lys176Ala and Glu313Ala mutant enzymes easily lost the flavin cofactor, yielding the corresponding apoproteins [238].This again demonstrates that changing a specific amino-acid residue can considerably influence the strength
of flavin binding.
In the near future, other recombinant-based methods such as the construction of fusion proteins will undoubtedly emerge for flavoprotein deflavination and reconstitution These procedures will be also valuable for the preparation of apo forms of other cofactor-containing proteins, specially when the apoprotein is relatively unstable in solution.
References
1 Spencer, R., Fisher, J.& Walsh, C.(1976) Preparation, char-acterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deazariboflavin 5¢-phosphate, and 5-deazariboflavin 5¢-diphosphate, 5¢ leads to 5¢-adenosine ester Biochemistry 15, 1043–1053
2 Karthikeyan, S., Zhou, Q., Mseeh, F., Grishin, N.V., Osterman, A.L.& Zhang, H.(2003) Crystal structure of human riboflavin kinase reveals a b barrel fold and a novel active site arch Structure 11, 265–273
3.Manstein, D.J.& Pai, E.F.(1986) Purification and character-ization of FAD synthetase from Brevibacterium ammoniagenes
J Biol Chem 261, 16169–16173
4.Massey, V.(2000) The chemical and biological versatility of riboflavin Biochem Soc Trans 28, 283–296
5.Palfey, B.A.& Massey, V.(1996) Flavin-dependent enzymes
In Comprehensive Biochemical Catalysis (Sinnott, M., ed.), pp 83–154.Academic Press, New York
Fig 4 Flavin binding in vanillyl-alcohol oxidase In native VAO, His61
(red) activates the neighbouring His422 (green) for covalent binding of
the FAD (yellow) cofactor.The aromatic ring of the inhibitor
iso-eugenol (blue) is situated parallel to the flavin isoalloxazine ring
Trang 106.Ghisla, S.& Massey, V.(1989) Mechanisms of
flavoprotein-catalyzed reactions Eur J Biochem 181, 1–17
7 Ballou, D.P., Williams, C.H & Coon, M.J (2002) Vincent
Massey (1926–2002) Trends Biochem Sci 27, 641–642
8 Mewies, M., McIntire, W.S & Scrutton, N.S (1998) Covalent
attachment of flavin adenine dinucleotide (FAD) and flavin
mononucleotide (FMN) to enzymes: The current state of affairs
Protein Sci 7, 7–20
9.Mathews, F.S.(1991) New flavoenzymes.Curr Opin Struct
Biol 1, 954–967
10.Fraaije, M.W.& Mattevi, A.(2000) Flavoenzymes: diverse
catalysts with recurrent features Trends Biochem Sci 25,
126–132
11.Dym, O.& Eisenberg, D.(2001) Sequence-structure analysis of
FAD-containing proteins Protein Sci 10, 1712–1728
12 Lee, Y.H., Nadaraia, S., Gu, D., Becker, D.F & Tanner, J.J
(2003) Structure of the proline dehydrogenase domain of the
multifunctional PutA flavoprotein Nat Struct Biol 10, 109–
114
13 Eggink, G , Engel, H , Vriend, G , Terpstra, P & Witholt, B
(1990) Rubredoxin reductase of Pseudomonas
oleovorans.Struc-tural relationship to other flavoprotein oxidoreductases based
on one NAD and two FAD fingerprints J Mol Biol 212,
135–142
14.Vallon, O.(2000) New sequence motifs in flavoproteins: evidence
for common ancestry and tools to predict structure Proteins 38,
95–114
15.Swindells, M.B.(1993) Classification of doubly wound
nucleo-tide binding topologies using automated loop searches Protein
Sci 2, 2146–2153
16 Eppink, M.H.M., Schreuder, H.A & van Berkel, W.J.H (1997)
Identification of a novel conserved sequence motif in flavoprotein
hydroxylases with a putative dual function in FAD/NAD (P) H
binding Protein Sci 6, 2454–2458
17 Fraaije, M W , Kamerbeek, N M , van Berkel, W J H &
Jans-sen, D.B (2002) Identification of a Baeyer-Villiger
mono-oxygenase sequence motif FEBS Lett 518, 43–47
18 Fraaije, M.W., van Berkel, W.J.H., Benen, J.A., Visser, J &
Mattevi, A.(1998) A novel oxidoreductase family sharing a
conserved FAD-binding domain Trends Biochem Sci 23,
206–207
19 Shima, S., Warkentin, E., Grabarse, W., Sordel, M., Wicke, M.,
Thauer, R.K.& Ermler, U.(2000) Structure of coenzyme F420
dependent methylenetetrahydromethanopterin reductase from
two methanogenic archaea J Mol Biol 300, 935–950
20 Sukumar, N , Xu, Y , Gatti, D L , Mitra, B & Mathews, F S
(2001) Structure of an active soluble mutant of the
membrane-associated (S)-mandelate dehydrogenase Biochemistry 40,
9870–9878
21 Vermilion, J.L & Coon, J.M (1978) Identification of the high
and low potential flavins of liver microsomal
NADPH-cyto-chrome P-450 reductase J Biol Chem 253, 8812–8819
22.Masters, B.S.S.(2000) Structural variations to accommodate
functional themes of the isoforms of nitric oxide synthases.In
Nitric Oxide (Ignarro, L., ed.), pp.91–104.Academic Press,
San Diego
23 Santolini, J., Adak, S., Curran, C.M & Stuehr, D.J (2001)
A kinetic simulation model that describes catalysis and
regula-tion in nitric-oxide synthase J Biol Chem 276, 1233–1243
24 Munro, A.W., Leys, D.G., McLean, K.J., Marshall, K.R., Ost,
T W , Daff, S , Miles, C S , Chapman, S K , Lysek, D A , Moser,
C.C., Page, C.C & Dutton, P.L (2002) P450 BM3: the very
model of a modern flavocytochrome Trends Biochem Sci 27,
250–257
25 Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D.,
Watkins, D , Heng, H H , Rommens, J M , Scherer, S W ,
Rosenblatt, D.S & Gravel, R.A (1998) Cloning and mapping
of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria Proc Natl Acad Sci USA 95, 3059–3064
26 Olteanu, H.& Banerjee, R.(2001) Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activa-tion J Biol Chem 276, 35558–35563
27.Eschenbrenner, M., Coves, J.& Fontecave, M.(1995) NADPH-sulfite reductase flavoprotein from Escherichia coli: contribution
to the flavin content and subunit interaction FEBS Lett 374, 82–84
28 Eschenbrenner, M., Coves, J.& Fontecave, M.(1995) The flavin reductase activity of the flavoprotein component of sulfite reductase from Escherichia coli.A new model for the protein structure J Biol Chem 270, 20550–20555
29 Ostrowski, J., Barber, M.J., Rueger, D.C., Miller, B.E., Siegel, L.M & Kredich, N.M (1989) Characterization of the flavo-protein moieties of NADPH-sulfite reductase from Salmonella typhimuriumand Escherichia coli J Biol Chem 264, 15796– 15808
30 Zeghouf, M., Fontecave, M., Macherel, D & Coves, J (1998) The flavoprotein component of the Escherichia coli sulfite reductase: expression, purification, and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors Biochemistry 37, 6114–6123
31 Champier, L , Sibille, N , Bersch, B , Brutscher, B , Blackledge, M.& Coves, J.(2002) Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase fla-vodoxin-like domain Biochemistry 41, 3770–3780
32 Binda, C , Bossi, R T , Wakatsuki, S , Arzt, S , Coda, A , Curti, B., Vanoni, M.A & Mattevi, A (2000) Cross-talk and ammonia channeling between active centers in the unexpected domain arrangement of glutamate synthase Struct Fold Des 8, 1299– 1308
33 van den Heuvel, R.H., Ferrari, D., Bossi, R.T., Ravasio, S., Curti, B., Vanoni, M.A., Florencio, F.J & Mattevi, A (2002) Structural studies on the synchronization of catalytic centers in glutamate synthase J Biol Chem 277, 24579–24583
34 Dobritzsch, D., Schneider, G., Schnackerz, K.D & Lindqvist, Y (2001) Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil EMBO J 20, 650–660
35 Husain, M.& Massey, V.(1978) Reversible resolution of flavo-proteins into apoflavo-proteins and free flavins Methods Enzymol 53, 429–437
36.Mu¨ller, F.& van Berkel, W.J.H.(1991) Methods used to reversibly resolve flavoproteins into the constituents apoflavoprotein and prosthetic group.In Chemistry and Biochemistry of Flavoenzymes (Mu¨ller, F., ed.), pp 261–274 CRC Press Inc, Boca Raton, FL
37.Lederer, F., Ru¨terjans, H.& Fleischmann, G.(1999) Flavopro-tein resolution and reconstitution.In FlavoproFlavopro-tein Protocols (Chapman, S.K & Reid, G.A., eds), pp 149–155 Humana Press Inc, Totower, New Jersey
38 Ghisla, S.& Massey, V.(1986) New flavins for old: artificial flavins as active site probes of flavoproteins Biochem J 239, 1–12
39.Edmondson, D.E.& Newton-Vinson, P.(2001) The covalent FAD of monoamine oxidase: structural and functional role and mechanism of the flavinylation reaction Antioxidant Redox Signal 3, 789–806
40 Mateo, P.L & Sturtevant, J.M (1977) Thermodynamics of the binding of flavin adenine dinucleotide toD-amino acid oxidase Biosystems 8, 247–253