Moreover, these structures show that OD2 of Asp171 accepts a proton from hydrogen peroxide in compound I formation, and that OD2 can swing to the appropriate position in response to the
Trang 1DyP may require the swinging movement of an aspartic acid residue
Toru Yoshida1, Hideaki Tsuge2, Hiroki Konno1, Toru Hisabori1and Yasushi Sugano1
1 R1-7 Chemical Resources Laboratory, Tokyo Institute of Technology, Japan
2 Department of Bioresources and Environmental Sciences, Kyoto Sangyo University, Japan
Introduction
Heme peroxidase, one of the best-studied and most
ubiquitous enzymes, oxidizes a variety of substrates,
including phenolic and azo compounds To date,
many heme peroxidases have been isolated from
vari-ous organisms, and the details of these can be found
in the high-quality PeroxiBase (http://peroxibase
toulouse.inra.fr/index.php) database [1] Heme
perox-idases are classified into six families: animal
peroxid-ases, nonanimal peroxidases, catalases, diheme
cytochrome c peroxidases, dye-decolorizing peroxidase
(DyP)-type peroxidases, and haloperoxidases The
cat-alytic cycle of peroxidases has been well studied [2],
and proceeds as follows:
Resting stateþ H2O2! compound I þ H2O
Compound Iþ AH2! compound II þ AH
Compound IIþ AH2! resting state þ AH þ H2O Combined with the above:
2AH2þ H2O2! 2H2Oþ 2AH
AH2 and AH• mean a substrate and a radical pro-duct, respectively In essence, enzymes that catalyze the above reaction are defined as peroxidases, even though details of the catalytic cycles or the nature of the
Keywords
catalytic mechanism; DyP; DyP-type
peroxidase; heme protein; swing
mechanism
Correspondence
Y Sugano, R1-7 Chemical Resources
Laboratory, Tokyo Institute of Technology,
4259 Nagatsuta, Midori-ku Yokohama
226-8503, Japan
Fax: +81 45 924 5268
Tel: +81 45 924 5235
E-mail: ysugano@res.titech.ac.jp
(Received 23 February 2011, revised 1 April
2011, accepted 4 May 2011)
doi:10.1111/j.1742-4658.2011.08161.x
The dye-decolorizing peroxidase (DyP)-type peroxidase family is a unique heme peroxidase family The primary and tertiary structures of this family are obviously different from those of other heme peroxidases However, the details of the structure–function relationships of this family remain poorly understood We show four high-resolution structures of DyP (EC 1.11.1.19), which is representative of this family: the native DyP (1.40 A˚), the D171N mutant DyP (1.42 A˚), the native DyP complexed with cyanide (1.45 A˚), and the D171N mutant DyP associated with cyanide (1.40 A˚) These structures contain four amino acids forming the binding pocket for hydrogen peroxide, and they are remarkably conserved in this family Moreover, these structures show that OD2 of Asp171 accepts a proton from hydrogen peroxide in compound I formation, and that OD2 can swing to the appropriate position in response to the ligand for heme iron On the basis of these results, we propose a swing mechanism in com-pound I formation When DyP reacts with hydrogen peroxide, OD2 swings towards an optimal position to accept the proton from hydrogen peroxide bound to the heme iron
Abbreviations
DyP, dye-decolorizing peroxidase; PDB, Protein Data Bank.
Trang 2intermediates obtained may differ from those of
stan-dard ubiquitous heme peroxidases Compound I
repre-sents an intermediate with an Fe4+oxoferryl center [3]
and a porphyrin cationic radical [4,5], whereas
com-pound II is obtained when one electron has been
removed from compound I The resulting AH• (an AH
radical) is further converted to various products via
non-enzymatic reactions (e.g radical coupling steps)
A great deal of information is available on the first
step of the cycle, i.e the generation of compound I
using H2O2 In the most popular model, a distal
histi-dine is essential, serving as an acid–base catalyst, and
an equally essential arginine supports compound I
for-mation [6] In other words, most heme peroxidases
have both a distal histidine and an essential arginine
[7] A simplified scheme of compound I formation,
according to many studies [8–10], is shown in Fig 1
The reaction starts with deprotonation of hydrogen
peroxide by histidine (an acid–base catalytic residue)
Although the proposed peroxidase–H2O2 complex
existing just before the formation of compound I (the
boxed state in Fig 1) has not been experimentally
demonstrated, it is believed that H2O2 must interact
with heme iron prior to compound I formation [8]
Deprotonation of H2O2 is extremely improbable,
because the pKaof this molecule is 11.6 [11] However,
H2O2 bound to heme iron (Fe3+) is estimated to have
a pKa of 3.2–4.0 [8] If this is indeed the case,
deproto-nation of H2O2 appears to be reasonable, and a
classi-cal reaction path (termed the Poulos–Kraut
mechanism) has been proposed [6]
DyP (EC 1.11.1.19) from the fungus Bjerkandera
ad-usta Dec 1 (formerly called Thanatephorus cucumeris
Dec 1) is a type of heme peroxidase, but also mediates
hydrolysis of anthraquinone rings, indicating that the
enzyme is bifunctional [12,13] DyP from B adusta
Dec 1 is a member of the DyP-type peroxidase family
[14], which is further subdivided into subfamilies A, B,
C, and D, according to PeroxiBase Research on DyP-type peroxidase family enzymes commenced only
15 years ago, much later than work on the more com-mon peroxidases However, in recent times, several major studies of DyP-type peroxidases have appeared [15–17] The DyP-type peroxidase AnaPX from the cyanobacterium Anabaena sp PCC 7120 (type D) shares some characteristics with DyP from B adusta Dec 1 [18] However, two other types of DyP-type per-oxidase, YcdB (type A) [19] and YfeX (type B) from Escherichia coli, have been reported to cooperatively capture iron [20] Surprisingly, the functions of YcdB and YfeX thus appear to have little to do with peroxi-dase activity These studies suggest that DyP-type per-oxidases represent a novel form of heme enzyme, expressing activities that are not confined to peroxidase action Moreover, such enzymes are widely distributed from bacteria to metazoa, indicating that the DyP-type peroxidase family is not exceptionally small, but is rather a sizeable grouping of proteins sharing type-unique characteristics Thus, a key cluster of proteins has evolved to play various roles in a variety of organ-isms [14] It is thus important to understand the vari-ous characteristics of DyPs In the present study, we focused on elucidation of the catalytic mechanism, employing tertiary structural analysis
Three X-ray crystal structures of members of the DyP-type peroxidase family, bound to heme or proto-porphyrin IX, have been deposited in the Protein Data Bank (PDB) YcdB (PDB ID 2WX7, 2.3 A˚ resolution)
is a type A enzyme; TyrA (PDB ID 2iiz, 2.3 A˚ resolu-tion) is a type B enzyme [21]; and DyP (PDB
ID 2D3Q, 2.96 A˚ resolution) is a type D enzyme [12] Surprisingly, the levels of primary structural identity between DyP (2D3Q) and the other enzymes are very low (< 5%), although the overall tertiary structures are very similar [14] One of the most interesting char-acteristics of DyP-type peroxidases is that the catalytic residue is not histidine but rather aspartic acid (Asp171 in DyP) [12] However, the details of struc-ture–function relationships in the DyP family remain poorly understood Here, we obtained four structures
of a DyP (type D) enzyme at 1.40–1.45 A˚ resolution: native DyP (native); the D171N mutant DyP (D171N), native DyP complexed with cyanide (CN) (native-CN), and the D171N mutant DyP associated with CN (D171N-CN), and identified the precise positions of Asp171 and associated residues These structures show that OD2 accepts a proton from H2O2 bound to the heme iron Nevertheless, in the native structure, OD2
is too far away to accept the proton from the H2O2
On the other hand, the four structures obviously show that OD2 is able to swing to the appropriate position
H
O
Fe3+
OH
H2O
Compound I Resting state
N NH
NH+
NH
N NH N
NH
–
O
Fig 1 Schematic diagram of the most popular mechanism
advanced to explain compound I formation by peroxidases
Struc-tures of the resting state, two deduced intermediates and
com-pound I are shown The black bars enclosing iron atoms represent
porphyrin rings The acid–base catalytic residue is indicated as the
imidazole group of histidine In compound I, the plus sign and
the black dot indicate when the porphyrin ring is a cationic radical.
The box indicates the region that is the focus of the present study.
Trang 3in response to ligand for heme iron On the basis of
these analyses, we propose a new mechanism for the
formation of compound I by DyP When H2O2 binds
to the heme iron of DyP, OD2 of the catalytic residue
Asp171 swings to a position that is optimal for
interac-tion with the H2O2
Results
Approach pathway and binding site of H2O2
In all heme peroxidases, H2O2is believed to bind to the
heme-distal side This means that an H2O2 approach
pathway from the enzyme molecular surface to the
heme-distal region must exist To date, no details of any
such pathway have been reported for DyP-type
peroxid-ases In the present study, we obtained a native structure
at 1.40-A˚ resolution (PDB ID 3AFV) This
high-resolu-tion structure showed much more precise details of
enzyme conformation and molecular surface
arrange-ment than were previously available [12] Heme was not
exposed to the molecular surface However, a large
cav-ity was evident towards the heme-distal side (Fig 2A),
and formed a small cylindrical pocket about 3 A˚ in both
diameter and height H2O2 appeared to fit into this
pocket In contrast, bulky substrates, such as
anthraqui-none, did not appear to bind into this pocket
Further-more, this region was surrounded by side chains of four
amino acids: Asp171 (the catalytic residue), Arg329,
Leu354, and Phe356 Overall, the results suggest that
H2O2may reach the heme-distal side by passing through
the cavity, and then bind and react (with heme) within
the pocket (Fig 2B)
Conservation of residues forming the binding
pocket for H2O2
Primary sequence identities among different types of
DyP-type peroxidases are, at most, 15% Indeed, this
value falls to < 5% when types A and D are compared Nevertheless, YcdB, TyrA, and DyP,
of types A, B, and D, respectively, have very similar b-barrel folds and a-helical structural regions As described above, we confirmed that the side chains of Asp171, Arg329, Leu354 and Phe356 form a binding pocket for H2O2 On the basis of X-ray crystal struc-tures and multiple sequence alignments among all DyP-type peroxidase family members (types A, B, C, and D), we confirmed conservation of the four residues that form binding pockets for H2O2 In YcdB (type A), the relevant residues are Asp200, Arg312, Leu331, and Phe333 In TyrA (type B), the residues are Asp151, Arg242, Leu255, and Phe257 Although BtDyP (type B) does not bind heme or protoporphy-rin IX (PDB ID 2gvk), the relevant residues in this protein seem to be Asp157, Arg245, Thr260, and Phe262 No X-ray crystal structure of a type C enzyme has been obtained However, multiple sequence align-ment of all DyP-type peroxidases in PeroxiBase showed that the four residues discussed above were remarkably conserved as compared with other residues (Fig S1) These results show that the b-barrel fold, the a-helical structural regions and the binding pocket for H2O2 are conserved in members of the DyP-type peroxidase family
D171N structure
We previously reported that the enzyme activity of the D171N was 1⁄ 3000th that of native [12] We obtained
a D171N structure at 1.42-A˚ resolution (PDB
ID 3MM1) (Fig 3), and compared this with the native structure at 1.40 A˚ resolution Both structures were superimposed on the heme plane and several rmsd val-ues were calculated (Table 1) The overall structure of the two enzymes was very similar The structures at the heme-distal side were also similar In both struc-tures, two water molecules were positioned in the
H2O2
D171
R329
F356 75°
R329
D171
F356 Heme plane H2 O2
3 Å
3 Å
Fig 2 Approach pathway and binding site of H2O2in DyP (A) The cutaway view indicates the molecular surface of the entire structure The black square indicates the heme plane The broken arrow in the large cavity shows the pathway taken by H2O2when it approaches the heme-distal side (B) Close-up views of an end of the cavity [circled in (A)] This region of the cavity forms a binding pocket for H 2 O 2 The broken arrow shows the approach pathway of H2O2 The pocket is delineated by double-headed arrows Four residues forming the pocket are shown in stick format.
Trang 4binding pocket of H2O2, and the positional relation-ships of the four residues forming the binding pocket were very similar OD1 of Asp171 (Asn171) formed hydrogen bonds with the amide nitrogen of Gly172 and NH1 of Arg329, but did not form a hydrogen bond with a water molecule in the binding pocket OD2 (ND2) did not form a hydrogen bond with the peptide chain, but formed a bond with a water mole-cule in the binding pocket No polar atoms that could form hydrogen bonds with OD2 (ND2) were noted in the peptide chain within 5.0 A˚ of OD2 (ND2) Excep-tionally, the positions of Asp171 and Asn171 seemed
to differ between the two structures In fact, the rmsd for Asp171(Asn171) was notably larger than that for other residues It is of particular interest that the rmsd for CA of Asp171(Asn171) was very small, but the rmsd for OD2 (ND2) was clearly large This probably results in alternation of proton acceptor and donor between the native and the D171N In the native tein, OD2 is the proton acceptor and W1227 the pro-ton donor In contrast, in the D171N mutant, ND2 is the proton donor and W1200 the proton acceptor These results strongly support the previous suggestion that Asp171 functions as a catalytic residue [12]
Coordination of CN
CN coordination was examined by spectroscopy and X-ray crystallography Because the speed of reaction between peroxidase and H2O2 is very high, the binding mode has not been experimentally demonstrated On the other hand, CN binds stably to the heme iron Actually, complexes of peroxidase with CN have been believed to mimic peroxidase–H2O2 complexes [22] Although the binding mode of the heme iron differs between H2O2 and CN, the position of the carbon atom of CN bound to the heme iron mimics the posi-tion of the proximal oxygen of H2O2 bound to the heme iron during compound I formation Therefore, the carbon atom position of the CN should provide basic information for understanding the interaction between OD2 of Asp171 and the proximal oxygen
of H2O2 bound to the heme iron in compound I formation
The addition of CN to native led to a shift in the Soret band from 406 nm to 421 nm, and created an additional absorption maximum at 535 nm with a shoulder at 565 nm (Fig 4) This change was similar
to that observed in horseradish peroxidase and Arthro-myces ramosus peroxidase upon binding of CN [2,23] The data suggest that CN bound to the heme iron of native, and that the electron state of the iron then changed from high-spin to low-spin We obtained a
Native
Native-CN
D171N
D171N-CN
D171
N171 G172
G172
G172
G172 R329
R329
R329
R329
F356 F356
CN CN
W1200 W1227
Fig 3 Structures at the heme-distal side of the native, D171N,
native-CN and D171N-CN enzymes Heme molecules are shown as
white sticks Blue spheres represent water molecules and W
means oxygen of water Broken lines between two atoms indicate
that the distance between these atoms is < 3.4 A ˚ The 2F o ) F c
electron density map at 1r is shown in pink for water molecules
and cyanide ions Brown circles represent the OD1 atom of
Asp171 or Asn171, and black circles represent the OD2 atom of
Asp171 or the ND2 atom of Asn171.
Table 1 Rmsd values between two structures.
Native and D171N
Native and native-CN
Native and D171N-CN
Asp171 (Asn171)
CG, OD1, OD2 (ND2)
atoms
Arg329
Leu354
Phe356
a The heme plane represents the 24 atoms of porphyrin.
Trang 5native-CN enzyme structure at 1.45 A˚ resolution (PDB
ID 3MM2) and one of D171N-CN at 1.40 A˚
resolu-tion (PDB ID 3MM3) (Fig 3) In both structures, CN
bound almost vertically to the heme plane, and the
nitrogen atom of CN formed a hydrogen bond with an
adjacent water molecule When the native-CN
struc-ture was compared with that of the native, by
superim-position in the heme plane, the rmsd for Asp171,
especially OD2, was apparently large (Table 1) When
D171N-CN was compared with the native by
superim-position on the heme plane, the rmsd for Asn171,
especially ND2, was again rather large Interestingly,
OD2 of Asp171 and ND2 of Asn171 formed hydrogen
bonds with different molecules In native CN, OD2
formed a bond with the water molecule adjacent to
CN In contrast, ND2 formed a hydrogen bond with
CN of D171N-CN
In the four structures obtained in the present study,
OD1 could not always form a hydrogen bond with a
molecule in the binding pocket of H2O2, but always
participated in formation of two hydrogen bonds with
the peptide chain In contrast, OD2 (ND2) could
always form a hydrogen bond with a molecule in the
binding pocket of H2O2, but could not always engage
in hydrogen bonding with the peptide chain These
results strongly suggest that OD2, rather than OD1,
accepts a proton from H2O2in the binding pocket
Discussion
Swinging of Asp171
It is important to note that OD2 of Asp171 in
native-CN and ND2 of Asn171 in D171N-native-CN formed
hydro-gen bonds with different molecules As a result, the
positions of OD2 and ND2 were very different This
appears to have been induced by variation in the
pro-tonation state of OD2 and ND2 In native-CN, OD2
is a proton acceptor at pH 6.0 The crystallization
con-dition was also at pH 6.0 Therefore, OD2 cannot form a hydrogen bond with CN, which is a proton acceptor, but can form such a bond with a water mole-cule adjacent to CN, which serves as a proton donor Thus, OD2 is shifted in position, in a direction away from the heme plane, as compared with the native On the other hand, in D171N-CN, ND2 is a proton donor
at pH 6.0 Therefore, ND2 can form a hydrogen bond with CN As a result, ND2 is shifted in position in a direction towards the heme plane, as compared with the native This suggests that OD2 can change position
in response to ligand status in the binding pocket This flexibility of OD2 seems to be associated with the fact that OD2 does not possess a polar atom that can form
a hydrogen bond Moreover, such flexibility was strongly supported by superimposition of the struc-tures of the native, D171N, native-CN and
D171N-CN enzymes in the heme plane (Fig 5) Because of differences in the chosen hydrogen bond partners, OD2 (ND2) seems to swing around OD1, by over 37 These results suggest that OD2 can swing in response
to ligand status
The catalytic residue Asp171 swings to form the compound I intermediate
At which position does OD2 accept a proton from
H2O2? Because an X-ray crystal structure at 1.4 A˚ reso-lution cannot show hydrogen atoms, we discuss this issue from the viewpoint of the distance between OD2 and the proximal oxygen We replace the carbon atom position of CN with a position of the proximal oxygen
Native Native-CN
5
Fig 4 UV–visible spectra of native and native-CN enzymes
Spec-tra from 450 nm to 700 nm are shown at five-fold magnification.
Solution conditions were 4 l M DyP in 25 m M citrate buffer (pH 5.5)
containing 0.5 M NaCl, with or without 100 m M KCN.
CN 2.05 A
37.0
OD1
OD2 (ND2)
D171 (N171)
D171 (N171)
OD1
OD2 (ND2)
60
Fig 5 Comparison of Asp171 (Asn171) locations between native (green), D171N (blue), native-CN (yellow) and D171N-CN (red) enzymes These four structures are superimposed on the heme planes Gray broken lines show the shortest distances between OD2 (ND2) and the carbon atom of CN for each structure The dis-tance between the CN carbon atom and the iron atom of heme is 2.05 A ˚ On the right, the relevant four residues are rotated by 60
to assist in an understanding of differences in residue positions.
Trang 6of H2O2as described in Results The distances between
OD2 (ND2) and the proximal oxygen are shown in
Fig 5 In the native and D171N-CN structures, the
dis-tances are 4.06 A˚ and 3.46 A˚, respectively We think
that this indicates a significant difference That is
because this difference is caused by the difference
between the positions of hydrogen bond partners of
OD2 in the native and ND2 in D171N-CN In the
native, the distance, 4.06 A˚, is too long to permit
reac-tion with H2O2 This shows that this position of OD2 in
the native is not appropriate for involvement in the
reac-tion In contrast, in D171N-CN, the distance, 3.46 A˚, is
less than in the native The position of OD2 in
D171N-CN is appropriate for involvement in the reaction Our
argument is illustrated in Fig 6A Thus, OD2 never
accepts a proton when in the position occupied by OD2
in the native, but does so when in the position occupied
by OD2 in D171N-CN (Fig 6A)
On the basis of the dual conclusions that OD2
accepts a proton when in the position occupied by
OD2 in D171N-CN, and that OD2 can swing from the
position occupied in the native to the position seen in
D171N-CN, we propose a swing mechanism for the
formation of compound I by DyP (Fig 6B) To accept
a proton, OD2 of Asp171 swings towards the position
occupied by OD2 in D171N-CN After compound I formation, OD2 of Asp171 returns to the position characteristic of the native The side chain of the cata-lytic residue of DyP is not located just above the heme iron, unlike in other heme peroxidases Moreover, the side chain of DyP is arranged in parallel rather than vertically to the heme plane This novel location and arrangement seems to produce the swing mechanism However, this swing mechanism may be needed in type C and D DyP-type peroxidases Nevertheless, we believe that this structural study paves the way to understanding the structure–function relationships of the DyP-type peroxidase family
Experimental procedures
Crystallization of native and D171N proteins Native and D171N proteins were purified with a modifica-tion of published procedures [12,24,25] Purified samples were deglycosylated by endoglycosidase H (Roche Diagnos-tics, Tokyo, Japan) Samples were loaded onto Superdex 75 columns (GE Healthcare Japan, Tokyo, Japan), and the de-glycosylated fractions were concentrated to 20 mgÆmL)1by ultrafiltration Crystallization was achieved with the
hang-Fig 6 The swinging mechanism of Asp171 (A) Interpretation of distances between Asp171 and a virtual proximal oxygen of H2O2in the native and D171N-CN The native and D171N-CN structures are superimposed on the heme planes Note that Asn171 of D171N-CN is shown as Asp171 To avoid misunderstanding, only the heme of the native is shown in white The position of the CN carbon mimics the position of the proximal oxygen of H2O2 Double-headed arrows show whether the distance between Asp171 and the virtual proximal oxy-gen is appropriate to permit reaction (B) Schematic diagram of the proposed mechanism of compound I formation by DyP Structures of the resting state, two deduced intermediates, and compound I are shown The two black bars enclosing an iron atom indicate the porphyrin ring Broken lines and arrows indicate hydrogen bonds and the swinging direction of the OD2 atom of Asp171, respectively In compound I, a plus sign and a black dot indicate when the porphyrin ring is a cationic radical.
Trang 7ing drop vapor diffusion method Drops containing 1 lL of
a 20 mgÆmL)1protein solution (0.1 m Mes at pH 6.0; 0.5 m
NaCl) and 1 lL of mother solution [0.1 m Mes at pH 6.0;
48% (w⁄ v) poly(ethylene glycol) 8000] were equilibrated
against 500 lL of reservoir solution [0.1 m Mes at pH 6.0;
0.25 m NaCl; 28% (w⁄ v) poly(ethylene glycol) 8000] at
278 K Hexagonal crystals appeared after 2–3 weeks Prior
to data collection, crystals were soaked briefly in a
cryopro-tective solution containing 0.1 m Mes at pH 6.0, 0.25 m
NaCl, 30% (w⁄ v) poly(ethylene glycol) 8000, and
25% (v⁄ v) glycerol, and flash frozen in liquid nitrogen
CN-complexed crystals of native and D171N
proteins
CN-complexed crystals were prepared by soaking native and
D171N proteins in a cryoprotective solution containing
0.1 m Mes at pH 6.0, 0.25 m NaCl, 30% (w⁄ v) poly(ethylene
glycol) 8000, 25% (w⁄ v) glycerol, and 120 mm KCN For
both crystal types, binding of CN appeared to be complete
within a few seconds, as assessed by visual monitoring of the
crystal color change from brown to red The crystals were
flash frozen in liquid nitrogen
X-ray data collection and structural refinement
Data collection from native, the D171N, native-CN and
D171N-CN was performed with a wavelength of 1.0 A˚ on
a beamline PF-AR NE3A or PF-AR NW12A instrument at
the Photon Factory (Tsukuba, Japan) Subsequent
proce-dures, including processing, scaling, and refinement, were
identical for all crystals Datasets were processed and scaled
with the hkl2000 program [26] Structures were solved with
the molecular replacement software of the ccp4 program
suite [27] (molrep), employing a 2.96-A˚-resolution structure
of DyP (PDB ID 2D3Q) as the starting point Iterative
refinement and model-building were subsequently
per-formed with refmac5 [28] and coot [29] Data collection
and refinement statistics are summarized in Table S1 For
native DyP, three datasets were collected, refined, and
superimposed on the heme plane The rmsd values for
Asp171 were very small in all three datasets (Fig S2;
Table S2) Two datasets were collected and refined for the
D171N, and superimposed on the heme plane The rmsd
values of Asn171 were rather large in both datasets This
was because the hydrogen bond between the protonated
ND2 of Asn171 and a water molecule in the binding pocket
was weak
UV–visible spectrophotometry (solution studies)
All spectra were obtained with a Shimadzu UV-2400 PC
spectrophotometer (Shimadzu Co., Kyoto, Japan) at 30C,
with a spectral bandwidth of 1.0 nm, employing cuvettes of
light path 1 cm Solution conditions were 4 lm DyP in
25 mm citrate buffer (pH 5.5) containing 0.5 m NaCl, with
or without 100 mm KCN
Acknowledgements
This research was undertaken with the assistance of the Photon Factory in KEK (proposal numbers 2010G011 and 2008G063) and was partly supported
by a Grant-in-Aid for Scientific Research (no 22570136) from the Japan Society for the Promo-tion of Sciences
References
1 Koua D, Cerutti L, Falquet L, Sigrist CJ, Theiler G, Hulo N & Dunand C (2009) PeroxiBase: a database with new tools for peroxidase family classification Nucleic Acids Res 37, D261–266
2 Dunford HB (1999) Heme Peroxidases Wiley, New York
3 Lang G, Spartalin K & Yonetani T (1976) Mo¨ssbauer spectroscopic study of compound ES of cytochrome c peroxidase Biochim Biophys Acta 451, 250–258
4 Browlett WR & Stillman MJ (1981) Evidence for heme
pi cation radical species in compound I of horseradish peroxidase and catalase Biochim Biophys Acta 660, 1–7
5 Kaneko Y, Tamura M & Yamazaki I (1980) Formation
of porphyrin pi cation radical in zinc-substituted horse-radish peroxidase Biochemistry 19, 5795–5799
6 Poulos TL & Kraut J (1980) The stereochemistry of peroxidase catalysis J Biol Chem 255, 8199–8205
7 Welinder KG (1992) Superfamily of plant, fungal and bacterial peroxidases Curr Opin Struct Biol 2, 388–393
8 Jones P & Dunford HB (2005) The mechanism of com-pound I formation revisited J Inorg Biochem 99, 2292– 2298
9 Raven EL (2003) Understanding functional diversity and substrate specificity in haem peroxidases: what can
we learn from ascorbate peroxidase? Nat Prod Rep 20, 367–381
10 Derat E, Shaik S, Rovira C, Vidosich P & Alfonso-Pri-eto M (2007) The effect of a water molecule on the mechanism of formation of compound 0 in horseradish peroxidase J Am Chem Soc 129, 6346–6347
11 Uri N & Evans MG (1949) The dissociation constant of hydrogen peroxide and the electron affinity of the HO2 radical Trans Faraday Soc 45, 224–230
12 Sugano Y, Muramatsu R, Ichiyanagi A, Sato T & Shoda M (2007) DyP, a unique dye-decolorizing peroxi-dase, represents a novel heme peroxidase family: Asp171 replaces the distal histidine of classical peroxid-ases J Biol Chem 282, 36652–36658
Trang 813 Sugano Y, Matsushima Y, Tsuchiya K, Aoki H, Hirai
M & Shoda M (2009) Degradation pathway of an
anthraquinone dye catalyzed by a unique peroxidase
DyP from Thanatephorus cucumeris Dec 1
Biodegradation 20, 433–440
14 Sugano Y (2009) DyP-type peroxidases comprise a
novel heme peroxidase family Cell Mol Life Sci 66,
1387–1403
15 Ogola HJ, Hashimoto N, Miyabe S, Ashida H,
Ishikawa T, Shibata H & Sawa Y (2010) Enhancement
of hydrogen peroxide stability of a novel Anabaena sp
DyP-type peroxidase by site-directed mutagenesis of
methionine residues Appl Microbiol Biotechnol 87,
1727–1736
16 Hofrichter M, Ullrich R, Pevyna MJ, Liers C & Lundell
T (2010) New and classic families of secreted fungal
heme peroxidases Appl Microbiol Biotechnol 87,
871–897
17 Liers C, Bobeth C, Pecyna M, Ullich R & Hofrichter
M (2010) DyP-like peroxidases of the jelly fungus
Auricularia auricular-judaeoxidize nonphenolic lignin
model compounds and high-redox potential dyes Appl
Microbiol Biotechnol 85, 1869–1879
18 Ogola HJ, Kamiike T, Hashimoto N, Ashida H,
Ishika-wa T, Shibata H & SaIshika-wa Y (2009) Molecular
character-ization of a novel peroxidase from the cyanobacterium
Anabaenasp strain PCC 7120 Appl Environ Microbiol
75, 7509–7518
19 Sturm A, Schierhorn A, Lindenstrauss U, Lilie H &
Bru¨ser T (2006) YcdB from Escherichia coli reveals a
novel class of Tat-dependently translocated
hemopro-teins J Biol Chem 281, 13972–13978
20 Le´toffe´ S, Heuck G, Delepelaire P, Lange N &
Wandersman C (2009) Bacteria capture iron from heme
by keeping tetrapyrrol skeleton intact Proc Natl Sci
Acad USA 106, 11719–11724
21 Zubieta C, Joseph R, Krishna SS, McMullan D,
Kapoor M, Axelrod HL, Miller MD, Abdubek P,
Acosta C, Astakhova T et al (2007) Identification and
structural characterization of heme binding in a novel
dye-decolorizing peroxidase, TyrA Proteins 69, 234–
243
22 Edwards SL & Poulos TL (1990) Ligand binding and
structural perturbations in cytochrome c peroxidase
J Biol Chem 265, 2588–2595
23 Fukuyama K, Kunishima N, Amada F, Kubota T &
Matsubara H (1995) Crystal structures of cyanide- and
triiodide-bound forms of Arthromyces ramosus peroxi-dase at different pH values Perturbations of active site residues and their implication in enzyme catalysis
J Biol Chem 270, 21884–21892
24 Sugano Y, Nakano R, Sasaki K & Shoda M (2000) Efficient heterologous expression in Aspergillus oryzae
of a unique dye-decolorizing peroxidase, DyP, of Geo-trichum candidumDec 1 Appl Environ Microbiol 66, 1754–1758
25 Saijo S, Sato T, Tanaka N, Ichiyanagi A, Sugano Y & Shoda M (2005) Precipitation diagram and optimization
of crystallization conditions at low ionic strength for deglycosylated dye-decolorizing peroxidase from a basidiomycete Acta Crystallogr F 61, 729–732
26 Otwinowski Z & Minor W (1997) Processing of x-ray diffraction data collected in oscillation mode Methods Enzymol 276, 307–326
27 Collaborative Computational Project Number 4 (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D 50, 760–763
28 Vagin A & Teplyakov A (2000) An approach to multi-copy search in molecular replacement Acta Crystallogr
D 56, 1622–1624
29 Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics Acta Crystallogr D 60, 2126–2132
Supporting information
The following supplementary material is available: Fig S1 Structure-based sequence alignments of YcdB, BtDyP, TyrA, and DyP
Fig S2 Comparison of the locations of Asp171 (Asn171) side chains among structures obtained with different datasets
Table S1 Data collection and refinement statistics Table S2 Rmsd values between two structures
This supplementary material can be found in the online version of this article
Please note: As a service to our authors and readers, this journal provides supporting information supplied
by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors