Functional characterization of an orphan cupin
protein from Burkholderia xenovorans reveals a
mononuclear nonheme Fe
2+
-dependent oxygenase
that cleaves b-diketones
Stefan Leitgeb, Grit D. Straganz and Bernd Nidetzky
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria
Introduction
In terms of their physiological functions, which include
enzymatic catalysis, ligand binding, and the role of
storage proteins, the cupins constitute one of the most
diverse superfamilies of proteins known. They have
been described from all three domains of life [1,2], and
usually occur as metalloproteins. Regardless of their
Keywords
cupin; nonheme iron; oxygenase; X-ray
absorption spectroscopy; b-diketone
cleavage
Correspondence
Bernd Nidetzky, Institute of Biotechnology
and Biochemical Engineering, Graz
University of Technology, Petersgasse 12 ⁄ I,
A-8010 Graz, Austria
Fax: +43 316 873 8434
Tel: +43 316 873 8400
E-mail: [email protected]
(Received 15 October 2008, revised 31 July
2009, accepted 17 August 2009)
doi:10.1111/j.1742-4658.2009.07308.x
Cupins constitute a large and widespread superfamily of b-barrel proteins

at estimated
distances of 2.04 A
˚
and 2.08 A
˚
, respectively. The three-histidine Fe
2+
site
of Bxe_A2876 is compared to the mononuclear nonheme Fe
2+
centers of
the structurally related cysteine dioxygenase and acireductone dioxygenase,
which also use a facial triad of histidines for binding of their metal cofac-
tor but promote entirely different substrate transformations.
Abbreviations
ARD, acireductone dioxygenase; CDO, cysteine dioxygenase; Dke1, b-diketone-cleaving dioxygenase; DLS, dynamic light scattering; EXAFS,
extended X-ray absorption fine structure; QDO, quercetin dioxygenase; RgCarb, Rubrivivax gelatinosus acetyl ⁄ propionyl-CoA carboxylase;
SOD, superoxide dismutase; XANES, X-ray absorption near-edge structure; XAS, X-ray absorption spectroscopy.
FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS 5983
low sequence homology, proteins classified as cupins
display a common double-stranded b-helix fold that
forms a core b-barrel. Two highly conserved histidine-
containing motifs separated by a variable intermotif
region provide the signature for the superfamily and
contribute the residues for metal binding [1,2]. A wide
range of catalytic functions, spanning primary enzyme
classes EC 1, EC 3, EC 4, and EC 5, have evolved in
cupin proteins [3–6]. Because the metal center usually
fulfils an essential role in catalysis by cupin enzymes,
there is the fundamental question of how the structures

further adds to the complexity of structure–function
relationships.
Fe
2+
cupins have recently attracted special atten-
tion because of the important roles that they play in
cell biology, such as DNA ⁄ RNA repair [15] and O
2
sensing [16]. Their ability to promote a wealth of
O
2
-dependent transformations has raised interest
among enzymologists and bioinorganic chemists. In
contrast to their catalytic versatility, the protein me-
tallocenters that bind the Fe
2+
display a remarkably
conserved structure [2,17–19]. A facial triad of two
histidines and one carboxylate residue (aspartate or
glutamate), exemplified by the metal centers of a
large class of 2-ketoglutarate-dependent oxygenases,
was long thought to form the canonical primary
coordination sphere for the Fe
2+
cofactor, as shown
in Fig. S1A [18].
With the expansion of the structural basis for Fe
2+
cupins, it has recently become clear that the original
two-motif structure of cupins, as in germin (Fig. S1B),

Burkholderia xenovorans through a database search in
which the cupin signature and the sequence of Dke1
from Acinetobacter johnsonii were used as queries.
The deduced primary structure of the novel cupin
protein Bxe_A2876 (UniProtKB: Q140Z1) and a
structural model derived from it suggested a cupin
protein featuring a three-histidine metal site. To
examine the unknown function of Bxe_A2876, we
performed a detailed biochemical characterization of
the recombinant protein produced in Escherichia coli.
A screening for O
2
-dependent enzyme activities elic-
ited by different combinations of metal and substrate
revealed that the Fe
2+
form of Bxe_A2876 was an
efficient catalyst of carbon–carbon bond cleavage in
b-diketone substrates. X-ray absorption spectroscopy
(XAS) was used to examine the coordination of Fe
2+
in the active site of the resting enzyme. The best fit
of the extended X-ray absorption fine structure (EX-
AFS) data indicated a five-coordinate or six-coordi-
nate Fe
2+
center that involves three nitrogen donors
from the histidine imidazole, one oxygen donor from
a carboxylate side chain, and one or two oxygen
donors from water. The Fe

ate vicinity of the metal center (Glu96, Thr105,
Met115, Phe117 and Leu121 in Bxe_A2876) are con-
served in the modeled structure relative to the experi-
mentally determined protein structures. It is therefore
interesting to note that the crystal structures of Dke1
and RgCarb were both solved for the respective Zn
2+
-
bound proteins. However, Dke1 requires Fe
2+
to be
active as a b-diketone-cleaving oxygenase. The first
coordination sphere of Fe
2+
could thus be different
from that of Zn
2+
seen in the enzyme structure (see
Discussion).
On an SDS ⁄ polyacrylamide gel of recombinant
Bxe_A2876 isolated from E. coli BL21(DE3), the puri-
fied protein migrated as a single band to the approxi-
mate position in the gel that was expected from its
predicted subunit size of 16 kDa (Fig. S3, lane 3).
Prior to purification and intein-tag cleavage, the bacte-
rial cell extract displayed a clear protein band of a size
corresponding to the  75 kDa mass for the fusion of
Bxe_A2876 and the IMPACT tag (Fig. S3, lane 2).
We used dynamic light scattering (DLS) to evaluate
the multiplicity of protomers in a preparation of

Fig. 2. CD spectrum of Bxe_A2876. Evaluation of the data was
performed with
DICHROWEB. The inset shows the distribution of
secondary structure elements. [h]
MRE
is the mean residual molar
ellipticity.
S. Leitgeb et al. b-Diketone-cleaving oxygenase from B. xenovorans
FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS 5985
functions of Bxe_A2876 on O
2
-dependent substrate
transformations catalyzed by members of the cupin
superfamily. Considering the structural similarity to
Dke1, special emphasis was placed on enzymatic reac-
tions involving Fe
2+
as cofactor. Reactions that could
have been promoted by a Zn
2+
protein were not inves-
tigated.
Preparations of Bxe_A2876 reconstituted with Fe
2+
or Cu
2+
were completely inactive against superoxide.
These proteins did not consume detectable amounts
of O
2

b-diketone substrate via oxidative carbon–carbon bond
cleavage to yield methylglyoxal and acetate.
Kinetic characterization of Fe
2+
Bxe_A2876
The activity of Bxe_A2876 in the O
2
-dependent con-
version of b-diketones was strictly dependent on the
Fe
2+
cofactor. We determined catalytic constants (k
cat
)
for different preparations of Bxe_A2876 whose frac-
tional occupancy with Fe
2+
varied between 0.1 and
0.9. Whereas the apparent value of k
cat
that was calcu-
lated from the V ⁄ [E] ratio (where V is the reaction rate
and [E] is the molar concentration of the 16 kDa
protein subunit) increased linearly with increasing
fractional saturation of the metal site in Bxe_A2876,
the k
cat
determined from the molar concentration of
Fe
2+

2+
.
To characterize substrate structural requirements
for the reaction catalyzed by Bxe_A2876, we tested a
series of b-diketones and related compounds in a two-
step assay. Enzyme substrates were first identified by
their ability to elicit O
2
consumption by Fe
2+
Bxe_A2876, and initial rate kinetic data were then
acquired by measuring spectroscopically the conversion
of the respective substrate. Previously reported molar
extinction coefficients for each active compound [27]
were confirmed and used in the determination of
reaction rates under conditions of apparent saturation
of the enzyme with the respective substrate. The
following k
cat
values were obtained: 0.4 s
)1
for 3,5-
heptanedione; 0.4 s
)1
for 2,4-octanedione; 0.2 s
)1
for
2,4-nonanedione; and 3.5 s
)1
for 2-acetylcyclohexa-

Scheme 1. Possible cleavage pathways of 1-phenyl-1,3-butanedione during enzymatic conversion by Bxe_A2876.
Table 1. Relative turnover numbers and cleavage ratios of 2,4-pentanedione and substituted variants. Activity measurements were per-
formed spectrophotometrically at 280 nm, where a decrease in absorbance reflects depletion of b-diketone substrate. Turnover numbers
were normalized using the k
cat
value for 2,4-pentanedione (0.8 s
)1
). Product analysis was performed by HPLC. The cleavage ratio is the ratio
of the concentrations of methylglyoxal (c
2
) and acetate (c
1
) formed upon conversion of unsymmetrical derivatives of 2,4-pentanedione. When
benzoylic substrates are used, the relevant ratio is that of phenylglyoxal (c
2
) and benzoate (c
1
). The preferred cleavage site in the respective
b-diketone substrate is indicated. The full set of experimental data used in the calculation of the cleavage ratio is shown in Table S4. NM,
not measured.
Structure Substrate Relative k
cat
Cleavage ratio, c
2
⁄ c
1
2,4-Pentanedione 1 1
1,1-Difluoro-2,4-pentanedione 2 · 10
)3
8.2

the presence of glucose and 2,4-pentanedione con-
tained a low level of specific activity (£ 5mU
mg
)1
protein). By contrast, no activity was measured
in cells grown on glucose alone. Addition of 2.0 mm
Fe
2+
to the assay strongly enhanced the enzyme activ-
ity by a factor of 10–50. Interestingly, upon comple-
mentation with Fe
2+
, differences in specific activity for
cells grown in the presence and absence of 2,4-pentan-
edione were essentially eliminated at a level of
 100 mU mg
)1
(Table S3). The specific activities
measured in B. xenovorans can be compared to a value
of  1600 mU mg
)1
for the purified recombinant
enzyme.
Characterization of the nonheme Fe
2+
center by
XAS
Figure 3A displays the XANES spectrum of
Bxe_A2876 around the Fe
2+

B
C
Fig. 3. X-ray absorption spectroscopy data for Bxe_A2876. (A)
Fe K-edge region in the XANES spectrum. (B) k
3
-weighted EXAFS
spectrum of Bxe_A2876 (solid line, black) overlaid by the fit model
of three histidines, one carboxylate, and one H
2
O (dotted line,
gray). v(k) is the EXAFS amplitude. See Table 2 for further details
of the fit. (C) Fourier transform (FT) of the EXAFS data. r is the
metal–ligand distance corrected for the phase shift.
b-Diketone-cleaving oxygenase from B. xenovorans S. Leitgeb et al.
5988 FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS
contained two or three nitrogen donors and three
oxygen donors at a distance of 2.00 A
˚
. Further refine-
ment was performed with excurv98, using various
models (Table S1) that incorporated histidine imidaz-
ole nitrogen atoms and different oxygen donor groups.
Separation of the single shell of scattering nitro-
gen ⁄ oxygen atoms into two shells (see center types 1
and 3 in Table S1) did not improve the goodness of fit
significantly, and gave differences in coordination
distance between the two shells (D  0.13–0.16 A
˚
) that
were generally at the limit of the resolution of the data

very well accounted for by center type 11, whereas it
was only poorly represented using center type 8. The
phase shift-corrected Fourier transform of the EXAFS
data is displayed in Figure 3C. Note that reasonable
Debye–Waller factors for all scattering atoms were
obtained using center type 11.
Discussion
Bxe_A2876 is an Fe
2+
-dependent oxygenase from
B. xenovorans that catalyzes the cleavage of carbon–
carbon bonds in b-diketone substrates. The enzyme is
not inducible by addition of b-diketone to the growth
medium. Cell extracts of B. xenovorans appear to
contain Bxe_A2876 largely in the inactive apo-form. It
is therefore possible that the enzyme recruits its redox-
active metal cofactor together with the substrate from
the solution complex of Fe
2+
and b-diketone, which is
known from the literature to be quite stable [33]. The
molecular and mechanistic properties of Bxe_A2876
are very similar to those of Dke1 (EC 1.13.11.50)
[28,34,35]. Evidence from XAS supports a five-coordi-
nate or six-coordinate Fe
2+
cofactor. Imidazole nitro-
gen atoms of His60, His62 and His102 and a
carboxylate oxygen atom, presumably contributed by
the side chain of Glu96, are suggested to function as

From the literature [28,34,35], the b-diketone bound
at the active site of Bxe_A2876 would seem to undergo
O
2
-dependent transformation into a C-3 peroxo inter-
mediate. Fe
2+
is expected to provide essential catalytic
assistance for this conversion. The low reactivity of
substrates harboring electron-withdrawing substituents
such as fluorine (Table 1) is explicable by a chemical
Scheme 2 Proposed reaction mechanism of Bxe_A2876.
S. Leitgeb et al. b-Diketone-cleaving oxygenase from B. xenovorans
FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS 5989
mechanism in which strong nucleophilic participation
of the substrate is required during the initial reduction
of O
2
[28,34–36].
Previous studies of Dke1 have also shown that elec-
tronic substituent effects on the distribution of prod-
ucts resulting from the cleavage of the b-diketone
substrate provide useful insights into the enzymatic
mechanism of carbon–carbon bond fission. Note, how-
ever, that the substituent effects governing the bond
cleavage steps are not the same as those controlling
the reactivity towards O
2
; hence the formation of the
proposed peroxo intermediate, which is rate-limiting

arising from the Fe
2+
in Bxe_A2876 are consistent
with five or six nitrogen ⁄ oxygen ligands of the bound
metal. Although X-ray absorption near-edge structure
(XANES) data favor a five-coordinate Fe
2+
, the pres-
ence of six donor groups, as in the related Fe
2+
sites
of CDO [27], human pirin [25,26], and gentisate 1,2-
dioxygenase [22,23], cannot be definitely ruled out. The
modeled structure of the nonheme metal site of
Bxe_A2876 (Fig. 1B) predicts that three nitrogen
donor ligands are contributed by the side chains of the
cupin triad of histidines, His60, His62, and His102.
This is in excellent agreement with the suggestion from
EXAFS analysis that three nitrogen atoms from the
histidine imidazole coordinate the Fe
2+
.
EXAFS data further suggest that the Fe
2+
center of
Bxe_A2876 does not involve a sulfur donor ligand,
again consistent with the model of the active site
(Fig. 1B), which has no candidate cysteine within a
realistic coordination distance from the likely position
of the Fe

pathways [37].
From the structure model of Bxe_A2876, the most
plausible candidate amino acid coordinating Fe
2+
would be Glu96. In a Zn
2+
-bound enzyme that was
completely inactive as a b-diketone-cleaving oxygenase
(data not shown) and therefore was not investigated
here, this glutamate could adopt an alternative, nonco-
ordinating, conformation that orients its carboxylate
side chain out of the metal center (Fig. 1B), as
observed for homologous glutamate residues in the
crystal structures of Zn
2+
-Dke1 and Zn
2+
-RgCarb.
The proposed Fe
2+
center (three histidines, one gluta-
mate, and one or two H
2
O) for Bxe_A2876 in the rest-
ing state is therefore novel among b-diketone-cleaving
oxygenases of the cupin protein superfamily, and
significantly advances our knowledge of the structural–
mechanistic basis for this group of enzymes. The opti-
mized metal–ligand distances (Table 2) compare very
favorably with data in the protein database, from

2+
center of
Bxe_A2876, as derived by the combination of mole-
cular modeling and XAS, is similar to related three-
histidine metal sites which were characterized by X-ray
crystallography [20,21,41,42], XAS [27,37], or a combi-
nation of the two methods [27,41]. The three-histidine
one-glutamate type of metal coordination was found
in high-resolution structures of resting state forms of
Cu
2+
-QDO [40], Ni
2+
-ARD [21], and Fe
2+
-pirin [25].
Metal coordination by a tetrad of three histidines
and one glutamate was likewise seen in other members
of the cupin protein superfamily, including oxalate
oxidase (germin) [6]. Interestingly, the position of
the glutamate ligand was not conserved in the amino
acid sequence, relative to the cupin core motifs that
contribute the three histidine ligands in each of these
proteins.
For QDO, XAS studies were performed with the
Cu
2+
enzyme [43]. XAS data for rat CDO in the rest-
ing state and in complex with l-cysteine were both
consistent with six nitrogen or oxygen donor ligands

3.15 A
˚
in the Fourier transform of the EXAFS spec-
trum of Bxe_A2876 (Fig. 3C) was not observed in the
corresponding spectra of CDO and ARD, suggesting
subtle differences in the coordination of Fe
2+
by
Bxe_A2876 as compared with the other two enzymes.
The active site of resting QDO was equally well
described by four or five ligands of Cu
2+
(three nitro-
gen donors from the histidine imidazole and one or
two oxygen donors) at an average distance of 2.00 A
˚
.
In the anaerobic complex of QDO and quercetin, the
Cu
2+
was five-coordinate, with three histidine nitrogen
donors and two oxygen donors in a single shell at
2.00 A
˚
[43].
Collectively, the XAS analysis for the Fe
2+
bound
to Bxe_A2876 makes an important contribution to the
characterization of the emerging three-histidine group

˚
) r
2
(A
˚
2
) E
0
(eV) R-factor (%) Fit index
1N⁄ O 5 2.02 0.013 )5.65 35.61 0.2207
2 His N 3 1.98 0.008
a
C 3.04 0.012
b
C 3.13 0.012
b
N 4.06 0.016
c
C 4.31 0.016
c
)6.57 16.65 0.0134
Glu O 1 2.04 0.008
a
C 3.31 0.012
b
O 4.28 0.012
b
C 3.83 0.012
b
H

(Waltham, MA, USA). All materials for genetic work were
obtained from New England Biolabs (Beverly, MA, USA)
and Fermentas International Inc. (Burlington, Canada).
Cultivation
Table S2 summarizes the different conditions in which the
B. xenovorans strain was incubated to examine growth and
the formation of oxygenase activity. All experiments were
performed in 80 mL shaken flasks at 30 °C, using an agita-
tion rate of 110 r.p.m. Bacteria obtained after growth for
48 h in medium B2 were used for inoculation of 250 mL of
medium to an initial attenuance at 600 nm of  0.4. Culti-
vation was continued for 48 h, and cells were harvested by
centrifugation (15 min, 4 °C, 4400 g). Crude cell extract
was prepared by lysis with B-Per reagent, following the
manufacturer’s protocol. The protein concentration was
determined using the bicinchoninic acid assay, and oxygen-
ase activity measurement was performed using the photo-
metric and HPLC assay described below. The activity of
the crude extract was expressed as mUÆmg
)1
protein. One
unit is defined as the amount of enzyme needed for the con-
version of 1 lmol acetylacetone min
)1
.
Cloning
The gene encoding Bxe_A2876 (accession number
gi:91782944) was amplified from genomic DNA of B. xenovo-
rans LB400 through a PCR with GAGCGG
CATATGGA

595 nm
) of 0.1 with
an overnight preculture of E. coli BL21(DE3). The strain
was incubated at 37 °C and 120 r.p.m. to a D
595 nm
of 0.6,
the temperature was reduced to 15 °C, and expression of the
target protein was initiated by addition of 250 lm isopropyl
thio-b-d-galactoside. Cells were harvested after approxi-
mately 20 h, resuspended in about the same volume of
20 mm Tris ⁄ HCl buffer (pH 7.5), and then disrupted by two
passages through a French press (American Instruments
Company, Silver Spring, MD, USA) operated at  8 MPa.
The cell-free extract was subsequently passed over a chi-
tin bead column (New England Biolabs, Beverly, MA,
USA), with a column volume of 15 mL. The column had
already been equilibrated with 10 column volumes of buf-
fer A (20 mm Tris ⁄ HCl, pH 7.5, 500 mm NaCl, 0.1%
Triton X). After the crude extract had been applied
( 650 mg of protein), the column was washed with 20
column volumes of buffer A, followed by three column
volumes of buffer B (20 mm Tris ⁄ HCl, pH 7.5, 500 mm
NaCl). Buffer B supplemented with 5 mm dithiothreitol
was employed to induce intein cleavage for 16 h at 15 °C.
The eluted protein was concentrated using Vivaspin concen-
trator tubes (M
r
cut-off of 10 000; Sartorius Stedim Biotech
S.A., Aubagne, France), and, finally, buffer was exchanged
in three cycles with 20 mm Tris ⁄ HCl (pH 7.5), with NAP

containing, typically, 17 mg of purified protein were pre-
pared in a mixture of 2 mL each of concentrated double-
distilled nitric acid, hydrochloric acid, and ultrapure water.
They were processed by microwave-assisted combustion
with a Multiwave 3000 microwave sample preparation
system (Anton Paar, Graz, Austria), using high-pressure
quartz vessels at 1400 W for 30 min [45]. Prior to measure-
ment, each sample was diluted 10-fold with water.
Reversible binding of Fe
2+
to Bxe_A2876
Metal-depleted Bxe_A2876 was prepared using Slide-A-
Lyzer 2K molecular weight cut-off dialysis cassettes (Pierce
Biotechnology, Rockford, IL, USA). About 1500 lLofa
solution containing 5 mg purified protein ⁄ mL was used.
Dialysis was performed at 4 °C for  48 h against 20 mm
Tris ⁄ HCl (pH 7.5), which was supplemented with 2 mm
EDTA for the first 12 h of the procedure. It was proven
that the protein preparation thus obtained did not contain
bound metal within the limits of detection of the analytical
methods used.
The apo-form of Bxe_A2876 was dissolved to a concen-
tration of 200 lm in 20 mm Tris ⁄ HCl (pH 7.5), and incu-
bated on ice in the presence of a 10-fold molar excess of
FeSO
4
over protein subunit of 16.1 kDa. The buffer and
the metal solutions used had been made micro-aerobic
(£ 15 lm dissolved O
2

592
= 35.5 mm
)1
Æcm
)1
) [46] was used to determine
the metal concentration.
O
2
-dependent conversion of b-diketones
Air-saturated buffer containing about 250 lm O
2
was used.
The concentration of the b-diketone substrate was 170 lm.
Reactions were performed in a total volume of 600 lL,
which contained 10 lL of enzyme, appropriately diluted to
measure the initial rate. The enzymatic rate was obtained
from the linear plot of substrate converted against the reac-
tion time (£ 2 min). Substrate consumption was monitored
from the decrease in absorbance at 280 nm, which reflects
breakdown of the b-diketone moiety. The following molar
extinction coefficients (e
280
) were used [28]: 2,4-pentanedi-
one, 2240 m
)1
Æcm
)1
; 3,5-heptanedione, 1200 m
)1

phate buffer (pH 7.8). The reaction mixture of 1 mL con-
tained 0.01 mm cytochrome c, 0.05 mm xanthine, and
0.005 U of xanthine oxidase from bovine milk (Sigma-
Aldrich, Gillingham, UK). Production of superoxide by
xanthine oxidase was followed indirectly by measuring the
increase in absorbance at 550 nm resulting from cyto-
chrome c being reduced by superoxide in the course of the
reaction. The principle of the assay is that the presence of a
SOD inhibits the reduction of cytochrome c. This was vali-
dated by adding a total of 2.5 U of commercial SOD from
bovine erythrocytes (Sigma-Aldrich, Gillingham, UK) to
S. Leitgeb et al. b-Diketone-cleaving oxygenase from B. xenovorans
FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS 5993
the assay. Experiments were performed in the presence and
absence of 0.1 mm EDTA, and yielded similar results.
Activity towards quercetin
The substrate solution contained 50 lm quercetin in 50 mm
Tris ⁄ HCl (pH 7.5), supplemented with 100 mm NaCl and
5% (v ⁄ v) dimethylsulfoxide. Purified Bxe_A2876 was added
in a final concentration of 4 lm metal sites. Reactions were
performed in the absence and presence of 2 mm CuSO
4
.
Conversion of quercetin was monitored from the decrease
in absorbance at 380 nm, using a reported molar extinction
coefficient of 18 500 m
)1
Æcm
)1
[47].

)1
in 20 mm
potassium phosphate buffer (pH 7.5), and analyzed. Tripli-
cate spectra obtained in the wavelength range 260–190 nm
were subsequently averaged and corrected by a blank
spectrum lacking enzyme before conversion of the CD
signal to mean residue ellipticity. Data were eventually
processed with the program dichroweb [48,49].
DLS
This was performed at 20 °C using an Fe
2+
-saturated prep-
aration of purified Bxe _A2876 dissolved to 1.0 mgÆmL
)1
in
20 mm potassium phosphate buffer (pH 7.5). Prior to mea-
surement, the protein sample was thoroughly centrifuged
(10 min, 16 000 g) to remove insoluble aggregates, and
45 lL of the supernatant was transferred to a cylindrical
quartz cuvette. Data were recorded using a Protein Solu-
tions DynaPro DLS instrument (Wyatt Technology Corpo-
ration, Santa Barbara, CA, USA), with the diode laser
wavelength and the sampling time set to 824.2 nm and
10 s, respectively. dynamics version 6 (Wyatt Technology
Corporation) was used to obtain hydrodynamic radius
distributions and the molecular mass distribution.
HPLC analysis of O
2
-dependent enzymatic
conversion of b-diketones

 1mm in 20 mm Tris ⁄ HCl (pH 7.5), supplemented with
20% glycerol (v ⁄ v) as cryoprotectant. It was loaded into
plastic holders of 1 mm thickness with polyimide windows,
and immediately flash-frozen in liquid nitrogen.
XAS measurements
These were carried out at 20 K, using the facilities at
EMBL Hamburg (DESY, EXAFS beamline D2, Hamburg,
Germany) with the DORIS storage ring operated at
4.5 GeV. An Si(111) double-crystal monochromator
scanned X-ray energies around the Fe K-edge (6.9–
7.85 keV). Harmonic rejection was achieved by a focusing
mirror (cut-off energy at 20.5 keV) and monochromator
detuning to 50% of its peak intensity. The X-ray absorp-
tion spectra were recorded as Fe K
a
fluorescence spectra
with a Canberra 13-element Ge solid-state detector. Data
reduction, such as background removal, normalization, and
extraction of the fine structure, was performed with kemp
b-Diketone-cleaving oxygenase from B. xenovorans S. Leitgeb et al.
5994 FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS
[50], assuming a threshold energy E
0,Fe
= 7120 eV. Sample
integrity during exposure to synchrotron radiation was
checked by monitoring the position and shape of the
absorption edge on sequential scans. No changes were
detectable.
Methods used in XANES and EXAFS analyses
winxas [51] was used to fit the pre-edge peak around

(Institute of Chemistry, University of Graz) in the
acquisition of CD and DLS data is gratefully acknowl-
edged. B. Kuczewski and H. Wiltsche (Institute of
Analytical Chemistry and Radiochemistry, Graz Uni-
versity of Technology) are thanked for metal analysis.
G. D. Straganz acknowledges support from the Aus-
trian Science Funds (FWF), project number P18828.
References
1 Khuri S, Bakker FT & Dunwell JM (2001) Phylogeny,
function, and evolution of the cupins, a structurally
conserved, functionally diverse superfamily of proteins.
Mol Biol Evol 18, 593–605.
2 Dunwell JM, Purvis A & Khuri S (2004) Cupins: the
most functionally diverse protein superfamily? Phyto-
chemistry 65, 7–17.
3 Cleasby A, Wonacott A, Skarzynski T, Hubbard RE,
Davies GJ, Proudfoot AE, Bernard AR, Payton MA &
Wells TN (1996) The x-ray crystal structure of phos-
phomannose isomerase from Candida albicans at 1.7 A
˚
resolution. Nat Struct Biol 3, 470–479.
4 Raymond S, Tocilj A, Ajamian E, Li Y, Hung MN,
Matte A & Cygler M (2005) Crystal structure of ureido-
glycolate hydrolase (AllA) from Escherichia coli
O157:H7. Proteins 61, 454–459.
5 Titus GP, Mueller HA, Burgner J, Rodriguez Cordoba
De S, Penalva MA & Timm DE (2000) Crystal struc-
ture of human homogentisate dioxygenase. Nat Struct
Biol 7, 542–546.
6 Anand R, Dorrestein PC, Kinsland C, Begley TP &

Ohlendorf DH (2004) Crystallographic comparison of
manganese- and iron-dependent homoprotocatechuate
2,3-dioxygenases. J Bacteriol 186, 1945–1958.
14 Emerson JP, Kovaleva EG, Farquhar ER, Lipscomb
JD & Que L Jr (2008) Swapping metals in Fe- and Mn-
dependent dioxygenases: evidence for oxygen activation
without a change in metal redox state. Proc Natl Acad
Sci USA 105, 7347–7352.
15 Yu B, Edstrom WC, Benach J, Hamuro Y, Weber PC,
Gibney BR & Hunt JF (2006) Crystal structures of
catalytic complexes of the oxidative DNA
⁄ RNA repair
enzyme AlkB. Nature 439, 879–884.
16 McDonough MA, Li V, Flashman E, Chowdhury R,
Mohr C, Lienard BM, Zondlo J, Oldham NJ, Clifton
IJ, Lewis J et al. (2006) Cellular oxygen sensing: crystal
S. Leitgeb et al. b-Diketone-cleaving oxygenase from B. xenovorans
FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS 5995
structure of hypoxia-inducible factor prolyl hydroxylase
(PHD2). Proc Natl Acad Sci USA 103, 9814–9819.
17 Joseph CA & Maroney MJ (2007) Cysteine Dioxy-
genase: Structure and Mechanism. Chem Commun 32,
3338–3349.
18 Clifton IJ, McDonough MA, Ehrismann D, Kershaw
NJ, Granatino N & Schofield CJ (2006) Structural
studies on 2-oxoglutarate oxygenases and related
double-stranded b-helix fold proteins. J Inorg Biochem
100, 644–669.
19 Hausinger RP (2004) Fe(II) ⁄ a-ketoglutarate-dependent
hydroxylases and related enzymes. Crit Rev Biochem

1491–1498.
26 Adams M & Jia Z (2005) Structural and biochemical
analysis reveal pirins to possess quercetinase activity.
J Biol Chem 280, 28675–28682.
27 Chai SC, Bruyere JR & Maroney MJ (2006) Probes of
the catalytic site of cysteine dioxygenase. J Biol Chem
281, 15774–15779.
28 Straganz GD, Glieder A, Brecker L, Ribbons DW &
Steiner W (2003) Acetylacetone-cleaving enzyme Dke1:
a novel C–C-bond-cleaving enzyme from Acinetobac-
ter johnsonii. Biochem J 369, 573–581.
29 Roe AL, Schneider DJ, Mayer RJ, Pyrz JW, Widom J
& Que L (1984) X-ray absorption spectroscopy of
iron-tyrosinate proteins. J Am Chem Soc 106,
1676–1681.
30 Randall CR, Shu L, Chiou Y-M, Hagen KS, Ito M,
Kitajima N, Lachicotte RJ, Zang Y & Que L (1995)
X-ray absorption pre-edge studies of high-spin iron(II)
complexes. Inorg Chem 34, 1036–1039.
31 Westre TE, Kennepohl P, DeWitt JG, Hedman B,
Hodgson KO & Solomon EI (1997) A multiplet analysis
of Fe K-edge 1s fi 3d pre-edge features of iron
complexes. J Am Chem Soc 119, 6297–6314.
32 Wellenreuther G & Meyer-Klaucke W (2007) Towards
a black-box for biological EXAFS data analysis – I.
Identification of zinc finger proteins. AIP Conf Proc
882, 322–324.
33 Fay DP, Nichols AR & Sutin N (1971) The kinetics
and mechanisms of the reactions of iron(III) with b-dik-
etones. The formation of monoacetylacetoatoiron(III)

40 Steiner RA, Kalk KH & Dijkstra BW (2002) Anaerobic
enzyme.substrate structures provide insight into the
reaction mechanism of the copper-dependent quercetin
2,3-dioxygenase. Proc Natl Acad Sci USA 99, 16625–
16630.
41 Simmons CR, Liu Q, Huang Q, Hao Q, Begley TP,
Karplus PA & Stipanuk MH (2006) Crystal structure of
mammalian cysteine dioxygenase. A novel mononuclear
iron center for cysteine thiol oxidation. J Biol Chem
281, 18723–18733.
42 Ye S, Wu XA, Wei L, Tang D, Sun P, Bartlam M &
Rao Z (2007) An insight into the mechanism of human
cysteine dioxygenase. Key roles of the thioether-bonded
tyrosine-cysteine cofactor. J Biol Chem 282, 3391–3402.
b-Diketone-cleaving oxygenase from B. xenovorans S. Leitgeb et al.
5996 FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS
43 Steiner RA, Meyer-Klaucke W & Dijkstra BW (2002)
Functional analysis of the copper-dependent quercetin
2,3-dioxygenase. 2. X-ray absorption studies of
native enzyme and anaerobic complexes with the
substrates quercetin and myricetin. Biochemistry 41,
7963–7968.
44 Sali A & Blundell TL (1993) Comparative protein
modelling by satisfaction of spatial restraints. J Mol
Biol 234, 779–815.
45 Flores EMM, Barin JS, Paniz JNG, Medeiros JA &
Knapp G (2004) Microwave-assisted sample combus-
tion: a technique for sample preparation in trace
element determination. Anal Chem 76, 3525–3529.
46 Johnson-Winters K, Purpero VM, Kavana M, Nelson

The following supplementary material is available:
Fig. S1. Structural comparison of nonheme metal-
dependent enzymes.
Fig. S2. Overlay of the homology model of Bxe_A2876
(white) with the template crystal structure of RgCarb
(RgCAR) (yellow) and the crystal structure of Acineto-
bacter johnsonii Dke1 (AjDKE) (green).
Fig. S3. Purification of Bxe_A2876 documented by
SDS ⁄ PAGE.
Fig. S4. Oxygen consumption during the conversion of
acetylacetone by Bxe_A2876.
Table S1. Various EXAFS model fits.
Table S2. Media composition for the cultivation of
Burkholderia xenovorans LB400.
Table S3. Growth and formation of oxygenase activity
by Burkholderia xenovorans LB400.
Table S4. Product distribution for the cleavage of
substituted diketone substrates.
This supplementary material can be found in the
online version of this article.
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should be addressed to the authors.
S. Leitgeb et al. b-Diketone-cleaving oxygenase from B. xenovorans
FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS 5997