Improved manganese-oxidizing activity of DypB, a peroxidase from a lignolytic bacterium

Article abstract of DOI:10.1021/cb300608x

DypB, a dye-decolorizing peroxidase from the lignolytic soil bacterium Rhodococcus jostii RHA1, catalyzes the peroxide-dependent oxidation of divalent manganese (Mn²⁺), albeit less efficiently than fungal manganese peroxidases. Substitution of Asn246, a distal heme residue, with alanine increased the enzyme's apparent k_cat and k_cat/K _m values for Mn²⁺ by 80- and 15-fold, respectively. A 2.2 A resolution X-ray crystal structure of the N246A variant revealed the Mn²⁺ to be bound within a pocket of acidic residues at the heme edge, reminiscent of the binding site in fungal manganese peroxidase and very different from that of another bacterial Mn²⁺-oxidizing peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variant's high K_m for Mn²⁺ (17 ± 2 mM). N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and its solvent-extracted fractions. Two of the major degradation products were identified as 2,6-dimethoxybenzoquinone and 4-hydroxy-3,5-dimethoxybenzaldehyde, respectively. These results highlight the potential of bacterial enzymes as biocatalysts to transform lignin.

Full text of DOI:10.1021/cb300608x

Articles
pubs.acs.org/acschemicalbiology
Improved Manganese-Oxidizing Activity of DypB, a Peroxidase from
a Lignolytic Bacterium
†
†
‡
‡
†
,†
Rahul Singh, Jason C. Grigg, Wei Qin, John F. Kadla, Michael E. P. Murphy, and Lindsay D. Eltis*
†
‡
Department of Microbiology and Immunology and Advanced Biomaterials Chemistry Laboratory, University of British Columbia,
Vancouver, British Columbia, Canada
*
S Supporting Information
ABSTRACT: DypB, a dye-decolorizing peroxidase from the
lignolytic soil bacterium Rhodococcus jostii RHA1, catalyzes the
2+
peroxide-dependent oxidation of divalent manganese (Mn ),
albeit less efficiently than fungal manganese peroxidases.
Substitution of Asn246, a distal heme residue, with alanine
increased the enzyme’s apparent k and k /Km values for
cat
cat
Mn2 by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray
+
2+
crystal structure of the N246A variant revealed the Mn to be
bound within a pocket of acidic residues at the heme edge,
reminiscent of the binding site in fungal manganese peroxidase and very different from that of another bacterial Mn -oxidizing
2
+
2
+
peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variant’s high K for Mn (17 ±
m
2
mM). N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and its solvent-extracted fractions.
Two of the major degradation products were identified as 2,6-dimethoxybenzoquinone and 4-hydroxy-3,5-dimethoxybenzalde-
hyde, respectively. These results highlight the potential of bacterial enzymes as biocatalysts to transform lignin.
1
0
ignin is a complex aromatic polymer that comprises
approximately 25% of the land-based biomass. It occurs in
proteobacteria. Nevertheless, the genes and the enzymes
involved in bacterial lignin degradation are not as well
characterized as their fungal counterparts, and lignin degrada-
tion products have not been identified. Bacterial enzymes that
have been implicated in lignin degradation include a lignin-type
L
tight association with cellulose and hemicellulose to form
lignocellulose, the rigid, recalcitrant material in woody plants.
Deconstructing this lignocellulose is critical to using biomass as
1
,2
9
a renewable source of energy and biomaterials. Lignin itself is
of burgeoning interest as a sustainable source of aromatic
peroxidase from Streptomyces viridisporus T7A, ALiP-P3, and
putative laccases and peroxidases from Enterobacter lignolyticus
SCF1. However, the best characterized are two actino-
bacterial dye-decolorizing peroxidases (DyP) that share ∼20%
amino acid sequence identity: DypB of Rhodococcus jostii
1
11
compounds, resins, and other biomaterials. Nevertheless,
lignocellulose-derived products are not economically viable
due in part to the energy-intensive processes used to
deconstruct biomass. Biocatalysts offer a greener, more
energy-efficient means to extract increased value from biomass.
The best characterized lignin-degrading enzymes are those
secreted by white rot fungi, such as Phanerochaete chrysospo-
12
13
RHA1 and DyP2 of Amycolatopsis sp. 75iv2. Both enzymes
catalyze the peroxide-dependent oxidation of Mn and the
2+
C −C bond cleavage of β-aryl ether lignin model com-
α
β
14,15
pounds.
DyP2 also possesses oxidase activity, catalyzing the
3
rium. These include lignin peroxidase (LiP), manganese
2+
O -dependent oxidation of Mn . Nevertheless, MnP from P.
4
5
2
peroxidase (MnP), versatile peroxidases, and laccases. LiP
and MnP are believed to oxidize small compounds or metals
that function as mediators for lignin oxidation. Thus, LiP
oxidizes veratryl alcohol, which in turn oxidizes nonphenolic
structures in lignin. For its part, MnP oxidizes Mn2+ to Mn ,
which is chelated by organic acids and oxidizes phenolic
15
2+
chrysosporium catalyzes the Mn oxidation ∼30,000- and 40-
14
fold more efficiently than DypB and DyP2, respectively.
2+
14
Structural data identified a Mn -binding site in DyP2 that is
located ∼15 Å from the heme, in contrast to the site at the
heme edge in MnP. The residues constituting the site in DyP2
are not conserved in other DyPs.
DyPs were first identified in the fungus Thanatephorus
cucumeris Dec1 for their ability to degrade anthraquinone
dyes. Genomic analyses have since revealed their occurrence
in a wide range of bacteria and fungi.
physiological role of DyPs remains unclear, the members of the
3+
3
+
structures present in the lignin. Oxidized Mn also generates
peroxy radicals, via lipid peroxidation, which oxidize the
6
,7
nonphenolic structures in lignin. Nevertheless, the industrial
applications of fungal enzymes have been limited by the
challenge of producing these post-translationally modified
16
12,17
Although the
8
proteins in commercially viable amounts. By contrast, bacterial
ligninases should be much easier to produce.
In the 30 years since the bacterial degradation of lignin was
Received: November 7, 2012
Accepted: January 10, 2013
Published: January 10, 2013
9
first reported, at least three classes of lignolytic bacteria have
been identified: actinomycetes, α-proteobacteria, and γ-
©
2013 American Chemical Society
700
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Articles
family catalyze a range of reactions of biotechnological
Table 1. Apparent Steady-State Kinetic Parameters of WT
DypB and Its Distal Residue Variants for Mn
2+
a
significance, including the oxidation of dyes, methoxylated
1
8
19
12,14,15
aromatic compounds, carotenoids, and lignin.
DyPs
cat (s−1)
kcat/K )
(× 10 M s−1
3
−1
20
enzyme
DypB
K
m
(mM)
k
m
belong to the CDE superfamily of heme peroxidases, sharing
a ferredoxin-type structural fold with chlorite dismutases, and
have been classified into four phylogenetically distinct
3.0 ± 0.3
2.8 ± 0.4
17 ± 2
ND
0.49 ± 0.02
1.6 ± 0.2
39 ± 3
ND
0.16 ± 0.01
0.57 ± 0.06
2.3 ± 0.1
ND
D153A
1
2,14,15
N246A
subfamilies:
DypB, DyP2, and DyPDec1 belong to the B,
D153A/N246A
C, and D subfamilies, respectively. DyPs share three conserved
residues in the heme-binding pocket: Asp153 and Arg244
b
DyP2
0.21 ± 0.03
0.060
24 ± 1
300
120 ± 20
5000
c
MnP
(
numbering of DypB from RHA1) on the distal face and
E39D
c
MnP
MnP
1.3
6.5
5
His226, the proximal ligand to the heme iron. The peroxidative
cycle of DyPs is similar to that of other peroxidases in that the
ferric enzyme reacts with H O to produce the highly oxidizing
intermediate Compound I ([Fe OPor ] ). However, the
catalytic machinery seems to function differently in the different
classes of DyPs since the distal aspartate is essential for
E39A
c
1.9
0.77
0.41
d
MnCcP
7.6
4.55
0.599
2
2
4
+
• +
a
Reactions were performed using 1 mM H O , 50 mM malonate, pH
2 2
b c
5
.5, at 25 °C. Data obtained from ref 14. Data obtained from ref 25.
d
Data obtained from ref 26. MnCcP has the W51F and W191F
2+
17
substitutions in addition to the engineered Mn -binding site.
peroxidase activity in DyPDec1, a D-type Dyp, but not in
DypB. Indeed, substitution of Asp153 had no effect on the
2
1
formation of Compound I in DypB but shortened its half-life by
for N246A (Figure 1, Table 1). By contrast, the D153A/N246A
double variant did not detectably oxidize Mn . This lack of
21
over 3 orders of magnitude to ∼0.13 s.
2+
Herein, we characterized the peroxidative oxidation of Mn2
+
activity is consistent with the very high K value of this variant
m
by DypB and investigated its utility for transforming lignin. The
roles of Asp153 and Asn246 in this reaction were studied, and
21
for H O (∼5 mM).
2
2
2+
The increased Mn -oxidation activities of N246A and
D153A presumably reflect the increased reactivity of Com-
pound I in these variants. More particularly, the k /K values
2+
the Mn -binding site of the enzyme was elucidated using X-ray
crystallography. Finally, the ability of a DypB variant to
transform solvent-fractionated hardwood kraft lignin (HKL)
was investigated, and lignin transformation products were
identified.
cat
m
of these variants for H O as well as their second order rate
2
2
constants with H O are very similar to those of WT DypB
2
2
5
−1 −1 21
(
∼10 M s ). By contrast, the half-life of Compound I in
these variants is ∼4,000-fold shorter, and their k values,
cat
21
2+
RESULTS AND DISCUSSION
Mn2+ Oxidation by DypB and Variants. We had
previously discovered that the distal heme pocket variants of
DypB, D153A and N246A, have higher peroxidase activity than
wild-type (WT) DypB using ABTS as a reducing substrate.
determined using either ABTS or Mn , are greater. While a
variety of factors can affect the reactivity of Compound I, one
obvious one is reduction potential. Indeed, the substitution of
Asn246 and Asp153, located 3.1 and 3.5 Å from the distal
solvent ligand of the heme iron, is expected to raise the redox
potential of the heme. By analogy, substitution of Val68 in
myoglobin, located on the distal face of the heme, with
aspartate and asparagine reduced the redox potential by 200
■
21
We therefore investigated the ability of these variants to
catalyze the peroxide-dependent oxidation of Mn2+ using a
steady-state kinetic assay monitoring the production of the
22
3+
and 80 mV, respectively. Nevertheless, further character-
ization of the WT and the variants is required to determine the
reason for increased reactivity of N246A. It is also unclear why
N246A appears to be a more efficient manganese peroxidase
than D153A. However, it is noted that the kcat value of D153A
Mn -malonate complex at 270 nm (Supplementary Figure 1).
The WT and variant DypBs each displayed Michaelis−Menten
2
+
behavior with respect to Mn concentration (e.g., Figure 1).
2+
Strikingly, the N246A and D153A variants oxidized Mn at
maximal rates (k ) that were 80- and 3-fold higher,
cat
21
is apparent since this variant’s K value for H O is 0.5 mM.
respectively, than WT DypB (Table 1). The K values for
m
2
2
m
2+
Mn2+ were comparable in WT and D153A, but ∼6-fold higher
Although the increase in the Mn -oxidation activity of
N246A versus the WT (80-fold increase in k ) is significantly
cat
greater than that in the ABTS-oxidation activity (1.7-fold
21
2+
increase in kcat), the specificity (k /K ) of N246A for Mn
cat
m
is 50- and 200-fold lower, respectively, than that of DyP2 and
MnP (Table 1). This presumably reflects the fact that DypB is
not a very efficient peroxidase as judged by its relatively low
5
−1 −1 21
second order rate constants with H O (∼10 M s ),
2
2
which is approximately 2 orders of magnitude lower than that
23
of efficient peroxidases, such as HRP.
2
+
Identification of a Mn -Binding Site. To characterize
2+
the interaction between DypB and Mn , the N246A variant
was crystallized in the presence of the divalent metal ion. X-ray
fluorescence scans of the crystals exhibited strong peaks for iron
2
+
and manganese (not shown). To identify the potential Mn -
binding site, a 2.2 Å resolution and a manganese K-edge data
set were collected from a single crystal (Table 2). The structure
of the N246A:Mn2+ complex was very similar (rmsd for Cα
atoms <0.3 Å) to WT DypB (PDB ID 3QNR). An anomalous
dispersion map generated from Mn2+ K-edge data revealed a
Figure 1. Steady-state kinetic analysis of Mn2+ oxidation by N246A.
Reactions contained 20 nM N246A and 1 mM H O in 50 mM
2
2
malonate, pH 5.5 at 25 °C. The solid line represents a best fit of the
Michaelis−Menten equation to the data using LEONORA.
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Articles
Table 2. X-ray Data Collection and Refinement Statistics for N246A:Mn2+ Complex
data set
high resolution
Data Collection
Mn K-edge
1.75858
wavelength (Å)
resolution range (Å)
space group
0.97530
46.7−2.2 (2.32−2.20)
P3 21
2
51.1−2.6 (2.74−2.60)
P3 21
2
unit cell dimensions (Å)
unique reflections
completeness (%)
average I/σI
a = b = 133.0, c = 159.5
83068
a = b = 133.1, c = 159.7
50809
99.8 (99.8)
8.4 (1.9)
99.9 (99.9)
8.3 (1.8)
redundancy
7.0 (7.1)
7.0 (7.0)
Rmerge
0.065 (0.376)
38.2
0.075 (0.430)
56.4
2
Wilson B (Å )
Refinement
0.170(0.196)
Rwork (Rfree
)
2
B-factors (Å )
all atoms
36.2
35.5
26.9
43.2
0.010
protein
heme
water
r.m.s.d. bond length (Å)
Ramachandran plot residues
in most-favorable region
in disallowed regions
PDB accession code
91.5
0.0
4HOV
large peak (∼14 σ) located in an acidic patch adjacent heme
Mn2+ (Figure 2). The second coordination sphere includes
hydrogen bonds between coordinating waters and Glu156,
propionate-D, composed of Glu156, Glu215, Thr231, and
24
Glu239 (Figure 2). The 2Fo − F electron density map
Glu215, Thr231, Glu239, and heme propionate-D.
c
2
+
The differences and similarities between the respective Mn -
binding sites of N246A, DyP2, and MnP are consistent with the
2
+
respective reactivities of these enzymes with Mn . First, the
enzyme with the lowest k value for Mn2+ (Table 1) is the one
cat
2+
in which the Mn -binding site is located furthest from the
2
+
24
heme. Thus, the Mn -binding sites of N246A and MnP are
located close to the heme edge and involve a heme propionate.
14
By contrast, that of DyP2 is ∼15 Å away from the heme. The
lower k value of DyP2 for Mn2+ is striking given that it is
cat
otherwise a more efficient peroxidase than DypB. The nature of
2+
the Mn -binding sites in the three enzymes also reflects their
2
+
relative K values for Mn (Table 1). Thus, N246A, whose
m
2+
Mn -binding site has no carboxylates in the first coordination
sphere of the metal ion, has a K value for Mn that is 300-
2+
2+
Figure 2. Active site and Mn -binding pocket of N246A. Residues
green) and heme (orange) are shown as sticks. The iron (dark
m
(
and 80-fold higher than that of MnP and DyP2, respectively.
Similarly, MnP, whose Mn -binding site comprises three
2+
2+
orange), solvent species (gray), and Mn (magenta) are shown as
2
+
spheres. The Mn -binding site is shown with an omit F − F map
o
c
glutamates and a heme propionate in the first coordination
(
gray mesh) contoured at 4.2σ and a Mn-anomalous map (red)
2+
sphere of the metal ion, has a K value for Mn lower than that
m
contoured at 8σ. Figures were made using PyMol.
of DyP2, which has two or three carboxylates in its site. The
importance of the first coordination sphere is further illustrated
2
5
by the effect of substituting Glu39 with Asp in MnP: this
indicates the presence of a hydrated metal coincident with the
2
+
2
+
relatively minor perturbation raised the enzyme’s K for Mn
peak in the anomalous map. Mn was modeled at 50%
occupancy to minimize residual difference density and by
comparison to anomalous peak heights for full-occupancy iron
m
25
by ∼20-fold (Table 1).
2+
The Mn -binding residues in DypB and DyP2 are not well
1
2,14
2+
conserved in DyPs,
suggesting that the binding of the
divalent metal ion may not be physiologically relevant.
Nevertheless, the structural models of the respective complexes
indicate that the Mn -oxidation activity of the DyPs could be
improved, either by engineering a Mn -binding site at the
(∼6.5 σ) and sulfur (∼7.7 σ) in the structure. The first Mn
coordination sphere was occupied exclusively by solvent
molecules with distances refined between 1.8 and 2.4 Å;
however, poorly defined density for the coordinating waters
suggests the solvent species are present in multiple
2+
2+
2
+
conformations and potentially occupy the Mn site in its
absence. The waters refined in distorted octahedral geometry,
but due to weak density at the sixth coordination site, only five
water molecules could be unambiguously modeled coordinating
heme edge in DyP2 or engineering a higher affinity site in
2+
N246A. Nevertheless, attempts to engineer Mn -binding sites
into peroxidases have been met with limited success. For
example, while cytochrome c peroxidase (CcP) has been
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ACS Chemical Biology
engineered (MnCcP) to bind Mn at a similar location as in
Articles
2
+
incubations with no enzyme developed significantly less
precipitate (<50% of initial lignin content) (Figure 3D).
Similar results were obtained using samples of softwood kraft
lignin and its solvent-extracted fractions (data not shown).
Chromatographic Analyses of Transformed HKL. The
soluble transformation products were characterized chromato-
graphically. Thus, the soluble portion of the reactions described
in the previous section was collected after 60 min of incubation
and purified using solid-phase extraction. Analysis of this extract
using reverse phase HPLC revealed that in enzyme-treated
samples a number of peaks with high retention times (tR)
2
6
2+
MnP, the kcat and k /K values of the most efficient Mn -
cat
m
2
4
oxidizing CcP variant were ∼10 - and 10 -fold lower,
respectively, than those of MnP from P. chrysosporium
Table 1). The limited success in improving the Mn -oxidizing
2
5,27
2+
(
activity of CcP suggests that other factors such as reduction
potential of the heme may also have to be engineered.
2
+
Mn -Dependent Transformation of Solvent-Fraction-
ated HKL by N246A. The ability of N246A to catalyze the
manganese-dependent transformation of lignin was investi-
gated. In these experiments, the variant was incubated with
hardwood kraft lignin (HKL), and each of 3 solvent-
fractionated samples (F1−F3). As will be described elsewhere
disappeared and a number of new peaks with low t appeared
(Figure 4). The new peaks had λmax between 280−310 nm,
R
(
Chowdhury, Wei, Kadla, unpublished), the average molecular
−1
weights of F1, F2, and F3 were 600, 1200, and 3200 g mol ,
respectively, similar to reported values. By comparison, the
28
average molecular weight of unfractionated HKL was 3300 g
−1
mol . Further, HKL and F3 contained 7.5% and ∼1%
carbohydrate, while F1 and F2 did not contain detectable
amounts. All transformations were performed in the presence
of MnSO (20 mM sodium malonate, pH 5.5, 15% DMSO).
4
Upon the addition of H O , the color of the reaction mixture
2
2
changed to burgundy within 10 min (Figure 3A). The
2+
Figure 4. Chromatographic analyses of the Mn -dependent trans-
formation of HKL with N246A. Reactions were performed for 60 min
essentially as described for Figure 3. The soluble products were
extracted and analyzed using reverse phase HPLC. The elution profiles
of reactions incubated with (red traces) and without (black traces)
N246A are shown.
Figure 3. Incubation of HKL and its fractions with N246A. Reaction
mixtures contained 0.25 mg ml−1 of a preparation of HKL, 100 nM
N246A, 20 mM MnSO , and 0.5 mM H O (20 mM sodium
4
2
2
malonate, pH 5.5, 15% DMSO) and were incubated at 30 °C. Panels A
and C show reactions after 10 and 60 min, respectively. Panels B and
D are the corresponding reactions performed in the absence of
enzyme. The lignin preparation used in each reaction is identified at
the top of each column.
consistent with them representing aromatic compounds, and
were of greater intensity in reactions performed with HKL-F1
than with other samples of HKL. The two most prominent new
peaks had t of ∼12 and 31 min and λ at 289 and 306 nm,
R
max
respectively. The peak that decreased most noticeably had a t
R
electronic absorbance spectrum of these samples revealed a
maximum at 495 nm, and the highest change in absorbance was
observed for samples containing HKL-F1 (data not shown).
Control reactions containing no enzyme did not show the same
color formation (Figure 3B). Incubation for 60 min resulted in
the formation of precipitate in most of the reactions (Figure
of ∼60 min and a λ at 270 nm. More generally, the spectral
max
features of this peak closely resemble those of HKL-F1 in
solution (data not shown). Time course studies showed that
the new peaks appeared with similar kinetics during the
transformation of the lignin and that the reaction was
essentially complete within 30 min (Supplementary Figure 2).
The more efficient transformation of HKL-F1 as compared to
the other lignin fractions is consistent with the former’s lower
3
C). The amount of precipitate accounted for up to 90% of the
lignin (dry weight) used in the reaction. In general, control
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Articles
average molecular weight and content of hemicellulose.
Further, the formation of precipitate during the enzymatic
reactions suggests that some polymerization also occurs, as has
been reported in MnP-catalyzed reactions. A comparison of
the transformation of HKL-F1 by DypB and N246A,
respectively, was also consistent with the kinetic data. Thus,
Figure 5C), and a database search revealed that its mass
spectrum (Figure 5, bottom panel) corresponded to that of the
trimethylsilyl derivative of 4-hydroxy-3,5-dimethoxybenzalde-
hyde (syringaldehyde). This was confirmed using authentic
29
syringaldehyde, whose t (on HPLC and GC (Supplementary
R
Figure 5D)), mass spectrum, and electronic absorption
spectrum matched those of the lignin-derived compound.
Using the authentic standards as control, the yields of 2,6-
DMBQ and syringaldehyde were determined, respectively, as
21 and 18 μg mg− HKL-F1.
the yield of the products with t of ∼12 and 31 min was ∼25%
R
and 2%, respectively, in the presence of DypB as compared to
N246A (Supplementary Figure 3).
1
Identification of the HKL-F1 Degradation Products.
Gas chromatography (GC) coupled mass spectrometry (MS)
was used to identify the compounds resulting from the N246A-
catalyzed transformation of HKL-F1. Consistent with the
HPLC analyses, the GC analyses of the soluble portion of
enzyme-treated HKL-F1 revealed the disappearance of several
peaks and the appearance of new ones as compared to controls
containing no enzyme (Supplementary Figure 4). Mass spectra
of the new peaks were consistent with the latter being lignin-
derived aromatic compounds (data not shown). The HKL-F1
transformation products eluting at 12 and 31 min, respectively,
were purified using semipreparative HPLC and analyzed using
The production of 2,6-DMBQ and syringaldehyde is
30
consistent with previous reports on MnP. Thus, MnP has
been shown to oxidize phenolic arylglycerol and aryl ether
lignin model compounds, producing 2,6-DMBQ and syringal-
dehyde. Further consistent with our results, syringaldehyde is
produced during the non-enzymatic oxidation of HKL as a
3
1,32
result of the cleavage of the Cα-Cβ bond.
The production
of benzoquinone structures, including 2,6-DMBQ, indicates the
32
cleavage of the C -Cα bond
and may also explain the
1
burgundy color that develops during incubation of HKL with
N246A. Finally, 2,6-DMBQ and syringaldehyde have been
produced during the oxidation of the nonphenolic lignin model
2-(2,6-dimethoxy-4-formylphenoxy)-1,3-dihydroxy-1-(4-
ethoxy-3-methoxyphenyl)-propane by laccase, a phenol oxidiz-
GC−MS. The product with t = 12 min on the analytical
R
reverse phase column had a tR = 10.8 min on the GC
(
(
Supplementary Figure 5A). The mass spectrum of this sample
Figure 5, top panel) was consistent with 2,6-dimethoxy-
33
ing enzyme, in the presence of 1-hydroxybenzotriazole. It
would be interesting to determine if N246A can catalyze similar
transformation of nonphenolic lignin in the presence of 1-
hydroxybenzotriazole.
Conclusions. This study shows that a bacterial peroxidase
2
+
can be engineered to improve its peroxide-dependent Mn
oxidation and lignin transformation capabilities. Although the
variant DypB is not as efficient as MnP or DyP2, the structural
data are consistent with the high K value of the enzyme for
m
Mn2+ and provide a basis for further optimizing the Mn -
2+
2
+
oxidizing activity of DypB. It is unclear whether Mn -oxidation
is physiologically relevant in DyPs, or whether DypB or DyP2
are even secreted. Nevertheless, there are DyP homologues that
have an alanine residue at the position equivalent to Asn246 in
DypB, including a DyP-type peroxidase from Streptomyces
viridochromogenes. Moreover, the residues involved in binding
Mn2+ in DypB are also conserved in the S. viridochromogenes
enzyme. Finally, the demonstration that N246A catalyzes the
12
2+
Mn -dependent transformation of lignin and that fractionated
lignins are transformed more efficiently highlights the potential
of bacterial ligninolytic enzymes as biocatalysts.
METHODS
■
Reagents and Chemicals. HPLC grade DMSO was from Alfa
Aesar. All other reagents and chemicals were purchased from SIGMA-
Aldrich, ACROS, MP Biomedicals, or Fisher and were used without
further purification. HKL was provided by FPInnovations (Pulp, Paper
and Bioproducts) and was solvent-fractionated essentially as will be
described elsewhere based on an established protocol. Water was
purified using a Barnstead NANO pure UV apparatus (Barnstead
International) to a resistivity of greater than 17 MΩ cm.
Figure 5. Structural characterization of transformation products
obtained from HKL-F1. The mass spectra of the purified products
eluting at t = 12 min (top) and t = 31 min (bottom) are shown.
28
R
R
Structures corresponding to the fragmentation patterns are shown in
inset.
Preparation of Recombinant DypB. Wild type DypB (WT),
D153A, N246A, and D153A/N246A were produced as apo-proteins
using pETDB, pETDBD153A, pETDBN246A, and pETDBD153A-
N246A, respectively, and were reconstituted with hemin chloride as
benzoquinone (2,6-DMBQ) in a catalogue search. Its identity
was confirmed using authentic 2,6-DMBQ, which had the same
tR (on HPLC and GC (Supplementary Figure 5B)), mass
spectrum, and electronic absorption spectrum as the lignin-
derived product. The product with t = 31 min on the analytical
reverse phase column was similarly purified and identified.
12,21
described previously.
Steady-State Kinetic Analysis. Apparent steady-state kinetic
2+
3+
R
parameters for Mn were determined by following Mn -malonate
12
formation using a previously described spectrophotometric assay.
Thus, this product had t = 13.8 min on GC (Supplementary
The assay was performed in 1 mL of 50 mM sodium malonate, pH 5.5
R
7
04
dx.doi.org/10.1021/cb300608x | ACS Chem. Biol. 2013, 8, 700−706

ACS Chemical Biology
Articles
at 25.0 ± 0.5 °C containing 0.2−40 mM MnSO and 20 nM N246A.
4
ASSOCIATED CONTENT
Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org
Accession Codes
■
The reaction was initiated by adding 1 mM H O and the initial rates
2
2
*
S
−1
−1 34
were monitored at 270 nm (ε270 = 11.9 mM cm ). 5Steady-state
3
kinetic equations were fit to the data using LEONORA.
X-ray Crystallography. Crystals of N246A were grown as
1
5
described previously. For co-crystallization of MnCl and N246A,
2
−1
protein samples (10 mg mL , 20 mM MOPS pH 7.5, 50 mM NaCl)
4HOV
were incubated with 167 mM MnCl at RT for 1 h. Drops were made
2
from 2 μL of protein-MnCl2 solution and 2 μL of well solution
consisting of 0.1 M sodium acetate trihydrate, pH 4.5, 3 M NaCl, and
crystals formed overnight. To prepare crystals for mounting, they were
briefly soaked in well solution supplemented to 16% glycerol and then
flash frozen by immersion in liquid nitrogen. Diffraction data were
collected on beamline 7-1 at the Stanford Synchrotron Radiation
Lightsource and are presented in Table 2. Data were processed using
AUTHOR INFORMATION
Corresponding Author
E-mail: leltis@mail.ubc.ca.
Notes
■
*
The authors declare no competing financial interest.
3
6,37
38
ACKNOWLEDGMENTS
Mosflm
and scaled with Scala. The structure was solved using
■
39
N246A (PDB ID: 3VEE) directly in Refmac
program suite and manually edited using Coot. Data are deposited
in the RCSB PDB under accession number 4HOV.
HPLC-Based Assay of Lignin Transformation. Mn -dependent
lignin transformation was routinely evaluated using reverse-phase
HPLC. Analyses were performed using a Waters 2695 Separations
module equipped with a Waters 2996 photodiode array detector.
from the CCP4
This work was supported by a Genome Canada Large-Scale
Research Project (to L.D.E. and J.K.), a Natural Sciences and
Engineering Research Council (NSERC) of Canada Strategic
Network (LignoWorks) (to L.D.E. and J.K.), and an NSERC
Discovery grants (to M.E.P.M.). Portions of this research were
carried out at the Stanford Synchrotron Radiation Lightsource,
a Directorate of SLAC National Accelerator Laboratory and an
Office of Science User Facility operated for the U.S.
Department of Energy Office of Science by Stanford University.
The SSRL Structural Molecular Biology Program is supported
by the DOE Office of Biological and Environmental Research
and by the National Institutes of Health, National Institute of
General Medical Sciences (including P41GM103393) and the
National Center for Research Resources (P41RR001209). The
contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of
NIGMS, NCRR, or NIH.
40
41
2+
−1
Reactions of 5 mL containing 0.25 mg mL preparations of HKL or
its fractions were incubated with 20 mM MnSO (20 mM sodium
4
malonate, pH 5.5, 15% DMSO) with or without 100 nM N246A. The
reaction was initiated by the addition of H O to 0.5 mM. After
2
2
incubation for 60 min, the precipitate was removed by centrifugation
4,000g × 30 min) and filtration (0.45 μm), washed 3 times with 1 mL
(
deionized H O, and dried under vacuum. The soluble portion was
2
acidified using formic acid (0.5% final concentration) and was then
extracted using solid-phase extraction (SPE). Briefly, the samples were
loaded onto a column packed with a C18 SPE matrix (Phenomenex)
equilibrated with 0.5% formic acid. The resin was washed using 5
column volumes of 0.5% formic acid. The reaction products were
eluted from the resin using 100% MeOH. The eluate was dried under
nitrogen. Air-dried samples were resuspended in 300 μL of 0.5%
formic acid, 20% methanol and filtered (0.2 μm). Samples of 90 μL
were injected onto a 150 mm × 3.00 mm C18 column (100 Å, 5 μm;
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Larger scale, 1 L reactions were performed using HKL-F1 to
maximize the product yield. This reaction was incubated for 3 h with
fresh MnSO , H O , and N246A added every hour. Following
6
(
4
2
2
incubation, the products were extracted as described above. The eluate
from the SPE resin was vacuum-dried and freeze-dried. Dried samples
were suspended in 50% MeOH, filtered (0.2 μm) and loaded (100 μL)
onto a 100 × 10 mm PFP(2) column (5 μm, 100 Å; Phenomenex)
(
−1
operated at 3 mL min . The products were eluted as described above.
(
2
(
Fractions eluting at t = 12 and 31 min were collected separately and
R
dried under nitrogen gas. The fraction eluting at tR = 12 min was
dissolved in 100 μL 30% acetonitrile, filtered (0.2 μm) and injected
onto the above-described C18 column equilibrated with 70 mM
−1
ammonium acetate, pH 8.5 and operated at a flow rate of 1 mL min .
The sample was eluted using the following gradient: 0% acetonitrile
for 2 min; 0 to 15% acetonitrile for 21 min; 15% to 100% acetonitrile
for 2 min.
Gas Chromatography−Mass Spectrometry (GC−MS) of HKL.
Samples were analyzed directly or were first derivatized using BSTFA
+
TMCS- (99:1). GC−MS was performed using an HP 6890 series GC
system fitted with an HP 5973 mass-selective detector and a 30 m ×
50 μm HP-5MS Agilent column. The operating conditions were TGC
2
(injector), 280 °C; TMS (ion source), 230 °C; oven time program
(
T0 ), 120 °C; T2 min, 120 °C; T30 min, 260 °C (heating rate 5 °C
min
−
1
min ); and T37 min, 260 °C.
7
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ACS Chemical Biology
Articles
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