The three-histidine Fe2+ site of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ centers of the structurally related cysteine dioxygenase and acireductone dioxygenase, which also u
Trang 1protein from Burkholderia xenovorans reveals a
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: bernd.nidetzky@tugraz.at
(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
in which a mononuclear metal site is both a conserved feature of the struc-ture and a source of functional diversity Metal-binding residues are con-tributed from two core motifs that provide the signature for the superfamily On the basis of conservation of this two-motif structure, we have identified an ORF in the genome of Burkholderia xenovorans that encodes a novel cupin protein (Bxe_A2876) of unknown function Recom-binant Bxe_A2876, as isolated from Escherichia coli cell extract, was a homotetramer in solution, and showed mixed fractional occupancy of its 16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc) Our quest for possible catalytic functions of Bxe_A2876 focused on Cu2+ and Fe2+ oxygenase activities known from related cupin enzymes Fe2+ elicited enzymatic catalysis of O2-dependent conversion of various b-dike-tone substrates via a nucleophilic mechanism of carbon–carbon bond cleavage Data from X-ray absorption spectroscopy (XAS) support a five-coordinate or six-coordinate Fe2+ center where the metal is bound by three imidazole nitrogen atoms at 1.98 A˚ Results of structure modeling studies suggest that His60, His62 and His102 are the coordinating residues
In the ‘best-fit’ model, one or two oxygens from water and a carboxylate oxygen (presumably from Glu96) are further ligands of Fe2+ at estimated distances of 2.04 A˚ and 2.08 A˚, respectively The three-histidine Fe2+ site
of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ 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.
Trang 2low 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
of cupin proteins determine metal-binding recognition
as well as reactivity in chemical transformations
Func-tional annotation of cupin proteins from sequence and
3D structural data alone is a challenging task [7], as
reflected by the recent addition of several new protein
structures (Protein Data Bank identifiers of selected
examples: 2PFW, 1VJ2, 2OZJ, 3ES1, 3EBR, 3FJS,
3ES4, 3D82, 3BCW, 3CEW, and 3CJX) to the
data-base without assignment of a putative function Metal
promiscuity in several cupin enzymes, including
super-oxide dismutase (SOD) (Fe2+, Mn2+) [8], acireductone
dioxygenase (ARD) (Ni2+, Fe2+) [9,10], quercetin
dioxygenase (QDO) (Cu2+, Fe2+) [11,12], and
homo-protocatechuate 2,3-dioxygenase (Mn2+, Fe2+) [13,14],
further adds to the complexity of structure–function
relationships
Fe2+ 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 O2
sensing [16] Their ability to promote a wealth of
O2-dependent transformations has raised interest
among enzymologists and bioinorganic chemists In
contrast to their catalytic versatility, the protein
me-tallocenters that bind the Fe2+ 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 Fe2+ cofactor, as shown
in Fig S1A [18]
With the expansion of the structural basis for Fe2+
cupins, it has recently become clear that the original
two-motif structure of cupins, as in germin (Fig S1B),
is also capable of forming a mononuclear nonheme
center for Fe2+, in which three histidines are
coordi-nated Structurally characterized cupin oxygenases
har-boring this alternative Fe2+ site are cysteine
dioxygenase (CDO) (Protein Data Bank identifier:
2ATF) [20], ARD (Protein Data Bank identifier:
1VR3) [21], QDO (Protein Data Bank identifiers:
1Y3T and 1JUH) [11,12], gentisate 1,2-dioxygenase
(Protein Data Bank identifiers: 2D40 and 3BU7) [22,23], and b-diketone-cleaving dioxygenase (Dke1) (Protein Data Bank identifier: 3BAL) [24] Pirins are nuclear proteins that also contain a three-histidine cen-ter for Fe2+ (Protein Data Bank identifiers: 1J1L and 1TQ5), and were recently shown to display QDO activ-ity [25,26]
Advances in our knowledge of structure–activity relationships for these and other three-histidine cen-ters of Fe2+ is currently limited by insufficient bio-chemical evidence, and would benefit from the characterization of novel cupin oxygenases of this group We identified an ORF in the genome of the polychlorinated biphenyl-degrading proteobacterium 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 O2-dependent enzyme activities elic-ited by different combinations of metal and substrate revealed that the Fe2+ 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 Fe2+
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 Fe2+ 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 Fe2+ center of b-diketone-cleaving oxygenase has not been previously character-ized structurally
Results
Structural properties of Bxe_A2876 Figure 1A shows a multiple alignment of the deduced primary structure of Bxe_A2876 with the sequences of Dke1 and a structurally characterized cupin protein from Rubrivivax gelatinosus PM1 (Protein Data Bank identifier: 2O1Q) that has been functionally annotated
as acetyl⁄ propionyl-CoA carboxylase (RgCarb) The three proteins share a high amount of sequence iden-tity (equal to or > 50%) and homology (equal to or
> 70%) A homology model of Bxe_A2876 was
Trang 3there-fore constructed, and the obtained fold was aligned
with the crystallographically determined structures of
the subunits of Dke1 (Protein Data Bank identifer:
3BAL) and RgCarb (Fig S2) Residue conservation in
the cupin two-motif structure of Bxe_A2876 suggests a
three-histidine metal center as in Dke1 and RgCarb
(Fig 1A) A close-up view of the nonheme metal site
in the structural model of Bxe_A2876 is given in
Fig 1B It supports the proposed mode of
coordina-tion with His60, His62 and His102 as metal ligands
Note that the coordinating histidines are contributed
from b-strands of the central cupin barrel, suggesting a
rather rigid metal-binding site Residues in the
immedi-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 Zn2+ -bound proteins However, Dke1 requires Fe2+ to be active as a b-diketone-cleaving oxygenase The first coordination sphere of Fe2+ could thus be different from that of Zn2+ 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 Bxe_A2876 as isolated, and results unambiguously showed the protein to be tetrameric The calculated molecular mass based on DLS data was 73 kDa, and corresponds reasonably with the predicted molecular mass of 64.4 kDa for the Bxe_A2876 homotetramer The relative distribution of secondary structure elements of Bxe_A2876 derived from CD spectroscopic data (Fig 2) agreed very well with the findings from the structure modeling studies
Metal-dependent reactivities of Bxe_A2876 The protein as isolated from E coli cell extracts showed mixed fractional occupancy of its 16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc) We focused the quest for possible enzymatic
A
B
Fig 1 Sequence analysis and structure modeling for Bxe_2876.
(A) Multiple sequence alignment of Bxe_A2876 with
Acinetobac-ter johnsonii Dke1 (AjDKE) and RgCarb (RgCAR) The sequence
alignment was performed with ALIGNX as a component of
VECTOR NTI 9.0.0, using standard settings Secondary structure
elements were manually assigned using the crystal structure of
RgCarb (Protein Data Bank: 2O1Q) H indicates a-helix, and –>
indicates b-strand Metal ligands are shown in bold, and conserved
residues are shown in italic (B) Predicted active site of Bxe_A2876
expanded 6 A ˚ around the metal center Figures were created with
PYMOL 0.99 [53].
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.
Trang 4functions of Bxe_A2876 on O2-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 Fe2+as cofactor Reactions that could
have been promoted by a Zn2+protein were not
inves-tigated
Preparations of Bxe_A2876 reconstituted with Fe2+
or Cu2+ were completely inactive against superoxide
These proteins did not consume detectable amounts
of O2 when one of the following substrates was
offered to a protein solution containing 7 lm metal
sites: xanthine in the presence of 2-ketoglutarate;
cat-echol; or quercetin However, when a series of
b-dike-tones were tested in combination with the Fe2+
protein (see below), the activity of Bxe_A2876
towards O2 was markedly stimulated as compared
with controls that contained apoprotein or lacked a
putative substrate The Cu2+ form of Bxe_A2876 did
not show activity under otherwise identical reaction
conditions
The activity of Fe2+ Bxe_A2876 with b-diketones
was characterized by comparing measurements of
con-sumption of O2 and substrate with the formation of
detectable products Figure S4 shows that conversion
of 2,4-pentanedione (5 mm) proceeded with depletion
of a molar equivalent of dissolved O2 HPLC analysis
of the products of the enzymatic transformation
revealed that Bxe_A2876 catalyzed breakdown of the
b-diketone substrate via oxidative carbon–carbon bond
cleavage to yield methylglyoxal and acetate
Kinetic characterization of Fe2+Bxe_A2876
The activity of Bxe_A2876 in the O2-dependent
con-version of b-diketones was strictly dependent on the
Fe2+cofactor We determined catalytic constants (kcat)
for different preparations of Bxe_A2876 whose
frac-tional occupancy with Fe2+ varied between 0.1 and
0.9 Whereas the apparent value of kcatthat 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 kcat determined from the molar concentration of
Fe2+-containing active sites was constant It had a
value of 0.8 s)1for air-saturated reaction conditions
at athmospheric pressure ( 250 lm O2) It was noted
that the dithiothreitol used in the intein cleavage step
of the purification protocol caused irreversible
inacti-vation of the purified enzyme (data not shown) We
therefore believe that dithiothreitol could be a source
of variation in the kcat of Bxe_A2876 preparations as
isolated However, attempts to replace dithiothreitol with b-mercaptoethanol (45 mm) or hydroxylamine (50 mm) in the purification proved fruitless The dura-tion of exposure of Bxe_A2876 to dithiothreitol was therefore kept as short as possible, and repeated cycles
of buffer exchange were used after the purification to carefully remove any of the dithiothreitol still present
in solution The reported kinetic data are for the of Bxe_A2876 exhibiting a kcat of 0.8 s)1 The Michaelis constant for 2,4-pentanedione was 5.1 lm (± 0.3 lm) and independent of the fractional occupancy of Bxe_A2876 with Fe2+
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 O2 consumption by Fe2+ 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 kcat 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-none By way of comparison, kcatvalues of Bxe_A2876 were lower, by about one order of magnitude, than the corresponding kcatvalues of Dke1 [28] Bxe_A2876 was inactive towards 3,3-dimethylpentanedione, 1,3-cyclohexanedione, and 4-methyl-2-oxovalerate, and these compounds are likewise not turned over by Dke1
Bond cleavage selectivity Unlike 2,4-pentanedione, whose symmetrical molecular structure dictates that its conversion by Bxe_A2876 can yield only a single pair of products, carbon–carbon bond cleavage in substrates harboring a different sub-stituent on either side of the central b-diketone moiety can proceed in one of two possible ways, each leading
to a characteristic pair of products Scheme 1 shows the possible reaction coordinates for 1-phenyl-1,3-butanedione We used HPLC analysis to determine the distribution of products obtained upon enzymatic con-version of a series of b-diketone substrates, which are listed in Tables 1 and S4 The results reveal that the bond cleavage selectivity of Bxe_A2876 was strongly influenced by the structural properties of the substitu-ents The turnover number of the enzyme also showed
a large substituent effect
Trang 5Scheme 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 (c2) and acetate (c1) formed upon conversion of unsymmetrical derivatives of 2,4-pentanedione When benzoylic substrates are used, the relevant ratio is that of phenylglyoxal (c2) and benzoate (c1) 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.
4,4-Difluoro-1-phenyl-1,3-butanedione 4 · 10)4 7.5
Trang 6b-Diketone-cleaving oxygenase activity in
B xenovorans
We examined growth and the formation of oxygenase
activity in B xenovorans using the media listed in
Table S2 The strain did not grow on 2,4-pentanedione
as the sole carbon source Growth was observed on a
mixed carbon source of glucose and 2,4-pentanedione
However, it was much lower than in the ‘glucose-only’
medium, suggesting that 2,4-pentanedione inhibits the
growth of the organism (Table S3)
Crude cell extracts of B xenovorans were analyzed
by SDS⁄ PAGE The distribution of protein bands on
the gel was not altered significantly in response to a
change in incubation conditions A protein of about
16 kDa was not abundant in the cell extracts (data not
shown) However, the b-diketone-cleaving oxygenase
activity displayed by isolated preparations of
recombi-nant Bxe_A2876 was clearly present in B xenovorans
Cell extracts obtained from bacteria incubated in
the presence of glucose and 2,4-pentanedione
con-tained a low level of specific activity (£ 5 mU
mg)1protein) By contrast, no activity was measured
in cells grown on glucose alone Addition of 2.0 mm
Fe2+to the assay strongly enhanced the enzyme
activ-ity by a factor of 10–50 Interestingly, upon
comple-mentation with Fe2+, 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 Fe2+center by
XAS
Figure 3A displays the XANES spectrum of
Bxe_A2876 around the Fe2+ absorption edge The
pre-edge feature of the spectrum at energies near
7113 eV reveals a forbidden 1s fi 3d electronic
tran-sition that, according to prior studies of nonheme
Fe2+ centers [29–31], is assigned to the mixing of the
4p orbital with the 3d orbital of the metal cofactor
Two important pieces of information can be gleaned
from the pre-edge peak First, occurrence of this
tran-sition implies distortion of the metal center from
per-fect octahedral geometry Second, the area associated
with the peak was previously shown to provide a
use-ful measure of the coordination number of the Fe2+
center [29–31] The value of (12 ± 1)· 10)2eV
there-fore indicates that the Fe2+ bound to Bxe_A2876 is
coordinated by a total of five ligand atoms
An initial estimation of the first coordination shell
of Fe2+ was made using abra [32] The average of the six best models lacked sulfur as Fe2+ ligand, and
A
B
C
Fig 3 X-ray absorption spectroscopy data for Bxe_A2876 (A)
Fe K-edge region in the XANES spectrum (B) k3-weighted EXAFS spectrum of Bxe_A2876 (solid line, black) overlaid by the fit model
of three histidines, one carboxylate, and one H2O (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.
Trang 7contained 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
( 0.14 A˚) Nitrogen and oxygen donor groups could
not be distinguished with the methods used However,
metal centers based on histidine imidazole nitrogen
donor groups clearly improved the goodness of fit, and
it was possible to identify a probable combination
of nitrogen and oxygen donor groups as well as the
corresponding metal–scatterer distances
Figure 3B shows the EXAFS data together with the
best theoretical fit that we obtained The suggestion
for the nonheme Fe2+center consists of three
imidaz-ole nitrogen atoms, one carboxylate oxygen atom from
either glutamate or aspartate, and one or two oxygen
atoms from water The comparison of fits provided by
metal center type 8 (three histidines, two H2O) and
type 11 (three histidines, one carboxylate, one H2O)
gives strong support to the idea of a carboxylate
oxy-gen ligand for the Fe2+ in Bxe_A2876 In particular,
the 3.1 A˚ scatterer peak in the EXAFS spectra was
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 Fe2+-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 Fe2+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-coordifive-coordi-nate Fe2+ 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 protein-derived ligands of the bound metal
Mechanistic deductions from biochemical data for Bxe_A2876
The proposed catalytic mechanism utilized by Bxe_A2876 in the O2-dependent conversion of 2,4-pen-tanedione is shown in Scheme 2 The results are consistent with participation of Fe2+as a redox-active cofactor in enzymatic catalysis of oxidative carbon– carbon bond cleavage However, a role of Fe2+ as an essential structural component of the active enzyme cannot be definitely ruled out on the basis of the data presented
As in Dke1, an important prerequisite for b-dike-tones to be accepted as substrates of Bxe_A2876 appears to be the ability to rearrange into a cis-b-keto–enol structure The required structure is not accessible, for chemical and steric reasons, respectively,
in 3,3-dimethylpentanedione and 1,3-cyclohexanedione Productive binding of 2,4-pentanedione and cognate b-diketones probably involves coordination to the
Fe2+ cofactor as cis-b-keto–enolates, as shown in Scheme 2
From the literature [28,34,35], the b-diketone bound
at the active site of Bxe_A2876 would seem to undergo
O2-dependent transformation into a C-3 peroxo inter-mediate Fe2+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.
Trang 8mechanism in which strong nucleophilic participation
of the substrate is required during the initial reduction
of O2[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 O2; hence the formation of the
proposed peroxo intermediate, which is rate-limiting
in the reaction catalyzed by Dke1 [35] Upon
intro-duction of the strongly electron-withdrawing
difluoro-methyl group, a marked shift in bond cleavage
specificity was observed as compared with the
corresponding specificity for the unsubstituted parent
substrate (Tables 1 and S4) The measured preference
for bond cleavage at the more electron-deficient
carbonyl carbon of the b-diketone moiety is consistent
with a nucleophilic mechanism of carbon–carbon bond
fission, where the C-3 peroxo intermediate undergoes
decomposition via a dioxetane (see [34,35] for a
detailed discussion) It is proposed in Scheme 2 that
the distal oxygen of the peroxidate performs an
intra-molecular attack on a neighboring carbonyl carbon,
preferably the one that harbors the relatively more
strongly electron-withdrawing substituent (e.g –CHF2
as compared with –CH3), to yield the dioxetane,
from which products are finally generated through
concerted C–C and O–O bond cleavage From the
evidence reported herein, as well as the previous
mech-anistic analysis for Dke1 [34], a Criegee rearrangement
mechanism of bond cleavage by Bxe_A2876 seems
unlikely
The three-histidine center of Fe2+in Bxe_A2876
Results of analysis of the X-ray absorption spectra
arising from the Fe2+ 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 Fe2+, the
pres-ence of six donor groups, as in the related Fe2+ 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 Fe2+
EXAFS data further suggest that the Fe2+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 Fe2+ There is, however, strong evidence from the EXAFS analysis that the Fe2+ cofactor is coordi-nated by an oxygen donor group derived from the carboxylate side chain of either a glutamate or an aspartate The apparent conflict of this finding with the absence of a coordinating carboxylate in the mod-eled metal center of Bxe_A2876 is reconciled by considering that the template structures used for homology modeling are for cupin proteins (Dke1, RgCarb) in their respective Zn2+-bound form Accom-modation of Fe2+ in the metallocenter may require a subtly different active site conformation from that employed for the binding of Zn2+ In a previous study
of ARD, it was shown that similar, but not identical, metal-binding modes are exploited for the coordination
of Fe2+ and Ni2+ cofactors However, the enzyme is active with both metal ions, despite different catalytic pathways [37]
From the structure model of Bxe_A2876, the most plausible candidate amino acid coordinating Fe2+ would be Glu96 In a Zn2+-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 Zn2+-Dke1 and Zn2+-RgCarb The proposed Fe2+ center (three histidines, one gluta-mate, and one or two H2O) for Bxe_A2876 in the rest-ing state is therefore novel among b-diketone-cleavrest-ing 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 which an average distance of 2.03 A˚ for Fe–N(His) was inferred [38], and a target distance of between 1.93 and 2.13 A˚ for Fe–O was obtained [39] Considering the proposed mode of substrate coordination by the
Fe2+of Bxe_A2876 (Scheme 2), it seems probable that metal ligation by protein side chains undergoes a change as result of binding of the b-diketone Mecha-nistically, a five-coordinate Fe2+ in the enzyme– substrate complex, as implied by Scheme 2, would leave one coordination site on the catalytic metal for reaction with O2 Work on QDO provides a relevant example, showing that the side chain of the glutamate
Trang 9participating in metal coordination in the free enzyme
rotates away upon accommodation of the substrate in
the active site [40] The possibility of Glu96 also
adopting a noncoordinating position in Bxe_A2876
has been mentioned
The model for the nonheme Fe2+ 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
Cu2+-QDO [40], Ni2+-ARD [21], and Fe2+-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
Cu2+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
being bound to the Fe2+ at average distances of
2.04 A˚ and 2.12 A˚, respectively [27] In resting ARD,
the Fe2+ was also six-coordinate and had two
nitro-gen(oxygen) donor ligands at an average distance of
1.90 A˚ and four nitrogen(oxygen) ligands at an
average distance of 2.06 A˚ Three of the four ligands
were reported to be consistent with histidine imidazole
side chains Upon formation of the ARD–acireductone
complex, the best fit of the EXAFS suggested three nitrogen(oxygen) donor ligands at an average distance
of 1.92 A˚ and three nitrogen(oxygen) donor ligands at
an average distance of 2.15 A˚, one of which was an imidazole side chain of histidine [37] Interestingly, the prominent second sphere feature at a distance of about 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 Fe2+ 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 Cu2+ (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
Cu2+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 Fe2+ bound
to Bxe_A2876 makes an important contribution to the characterization of the emerging three-histidine group
of nonheme Fe2+ centers The results obtained are in useful agreement overall with suggestions from struc-ture modeling studies of Bxe_A2876 The XAS data indicate that binding of Fe2+ may require a different active site conformation than binding of Zn2+ Flexi-bility of the conserved Glu96 could have a role in determining metal-binding selectivity An immediate question that arises upon comparison of the XAS data for Bxe_A2876, CDO and ARD pertains to the rela-tionship between coordination of the catalytic metal and reactivity in different O2-dependent transforma-tions Examination of the function of noncoordinating
Table 2 Proposed ligand environment of the iron bound in the active site of Bxe_A2876 in the resting state, as derived from EXAFS data analysis (fit 2) The ‘best-fit’ model (fit 2) is compared with one of the initial models considered (fit 1) See Table S1 for further details of EXAFS data analysis N is the number of ligands, r is the distance to the central iron atom, and r 2 is the Debye–Waller factor The k-range is 2–13 A˚)1 See Table S1 for further details of EXAFS data analysis.
a,b,c
The Debye-Waller factors were grouped and refined in batches (a, b and c).
Trang 10residues in and around the active site may provide the
answer Systematic comparison of biochemical
infor-mation with structural evidence for different
three-histidine cupin enzymes will hopefully lead to the
identification of fingerprint regions that determine
metal-binding selectivity and catalytic activity (see
the recent work on ARD [37]) Within the cupin
super-family of proteins, structure-based distinction between
Fe2+and Mn2+forms of SOD provides an interesting
example [8]
Experimental procedures
Materials
B xenovoransLB400 was obtained from the Belgian
Co-ordinated Collections of Micro-organisms (BCCM,
Gent, Belgium), deposited under accession number
LMG 21463 It was grown using a protocol supplied by
BCCM All chemicals were purchased from Sigma-Aldrich
(Gillingham, UK) in the highest available purity B-Per and
the bicinchoninic acid assay were from Thermo Scientific
(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 xenovoransstrain was incubated to examine growth and
the formation of oxygenase activity All experiments were
performed in 80 mL shaken flasks at 30C, 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, 4C, 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-ransLB400 through a PCR with GAGCGGCATATGGA
AATCAAACCGAAGGTTCGCGA and GAGCGGCATA
TGGAAATCAAACCGAAGGTTCGCGA as the forward
and reverse oligonucleotide primers, respectively The
primers were designed to introduce restriction sites (under-lined) for NdeI and SapI, respectively The amplified gene and a pTYB1 plasmid vector were digested with NdeI and SapI in a one-pot reaction at 37C for 1 h After purification
of the DNA by precipitation with ethanol, the gene was ligated with the pTYB1 vector using T4 DNA ligase in an overnight reaction at room temperature Following degrada-tion of empty plasmid by XhoI (37C, 1 h), the ligation mixture was transformed into E coli TOP10 cells by electro-poration Cells were subsequently transferred to LB–ampicil-lin plates Single colonies were picked, and plasmids were isolated with a QIAprep spin Miniprep Kit from Qiagen (Hilden, Germany) Positive clones were selected by restric-tion analysis with ClaI and sequenced (MWG Biotech, Ebersberg, Germany) The pTYB1 vector harboring the target gene was transferred into the expression strain
E coliBL21(DE3) It encodes a chimeric form of Bxe_A2876 that has the IMPACT tag fused to the authentic C-terminal Gly145
Expression and purification Recombinant protein was produced by cultivating the expression strain in shaking flasks containing LB medium supplemented with 100 lgÆmL)1ampicillin The media were inoculated to an attenuance at 595 nm (D595 nm) of 0.1 with
an overnight preculture of E coli BL21(DE3) The strain was incubated at 37C and 120 r.p.m to a D595 nmof 0.6, the temperature was reduced to 15C, 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 15C The eluted protein was concentrated using Vivaspin concen-trator tubes (Mrcut-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 columns (GE Healthcare, Chalfont St Giles, UK) Purifica-tion was checked by SDS⁄ PAGE Protein solutions ( 5 mgÆmL)1) were stored in 100 lL aliquots at )20 C until further use Repeated freeze–thaw cycles of the protein stock solution were avoided