Retuning rieske-type oxygenases to expand substrate range

Article abstract of DOI:10.1074/jbc.M111.255174

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE_p4, a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE_LB400 by the double substitution T335A/F336M, and BphAE_RR41, obtained by changing Asn³³⁸, Ile³⁴¹, and Leu⁴⁰⁹ of BphAE _p4 to Gln³³⁸, Val³⁴¹, and Phe⁴⁰⁹, metabolize dibenzofuran two and three times faster than BphAE_LB400, respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE_LB400 showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reactioncritical atoms or groups.

Full text of DOI:10.1074/jbc.M111.255174

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 31, pp. 27612–27621, August 5, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Retuning Rieske-type Oxygenases to Expand Substrate
Range*
Received for publication, April 27, 2011, and in revised form, May 25, 2011 Published, JBC Papers in Press, June 8, 2011, DOI 10.1074/jbc.M111.255174
Mahmood Mohammadi‡1, Jean-Franc¸ois Viger‡1, Pravindra Kumar§¶1, Diane Barriault‡, Jeffrey T. Bolin§,
and Michel Sylvestre‡2
From the ‡Institut National de la Recherche Scientifique-Institut Armand-Frappier, Laval, Quebec H7V 1B7, Canada, the
§Department of Biological Sciences and Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, and the
¶Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, India
Rieske-type oxygenases are promising biocatalysts for the
destruction of persistent pollutants or for the synthesis of fine
chemicals. In this work, we explored pathways through which
Rieske-type oxygenases evolve to expand their substrate range.
BphAEp4, a variant biphenyl dioxygenase generated from Burk-
holderia xenovorans LB400 BphAELB400 by the double substitu-
tion T335A/F336M, and BphAERR41, obtained by changing
Asn338, Ile341, and Leu409 of BphAEp4 to Gln338, Val341, and
Phe409, metabolize dibenzofuran two and three times faster than
BphAELB400, respectively. Steady-state kinetic measurements of
single- and multiple-substitution mutants of BphAELB400
showed that the single T335A and the double N338Q/L409F
substitutions contribute significantly to enhanced catalytic
activity toward dibenzofuran. Analysis of crystal structures
showed that the T335A substitution relieves constraints on a
segment lining the catalytic cavity, allowing a significant dis-
placement in response to dibenzofuran binding. The combined
N338Q/L409F substitutions alter substrate-induced conforma-
tional changes of protein groups involved in subunit assembly
and in the chemical steps of the reaction. This suggests a respon-
sive induced fit mechanism that retunes the alignment of pro-
tein atoms involved in the chemical steps of the reaction. These
enzymes can thus expand their substrate range through muta-
tions that alter the constraints or plasticity of the catalytic cavity
to accommodate new substrates or that alter the induced fit
mechanism required to achieve proper alignment of reaction-
critical atoms or groups.
zofurans (6, 7), or to produce chiral arene cis-dihydrodiols of
interest in enantioselective syntheses for the manufacture of
fine chemicals (8–10). Biphenyl dioxygenase, one of the most
extensively studied ROs, catalyzes the first reaction of the bac-
terial biphenyl catabolic pathway. Biphenyl dioxygenase has
three components: the iron-sulfur oxygenase (hereinafter
referred to as BphAE), a heterohexamer comprised of three ␣
(Mr ϭ 51,000) and three ␤ (Mr ϭ 22,000) subunits; the ferre-
doxin (BphF, Mr ϭ 12,000); and the ferredoxin reductase
(BphG, Mr ϭ 43,000). The encoding genes for Burkholderia
xenovorans LB400 (11), which is the best polychlorinated
biphenyl degrader of natural origin, are bphA (BphAELB400
␣
subunit), bphE (BphAELB400 ␤ subunit), bphF (BphFLB400), and
bphG (BphGLB400) (see Fig. 1).
BphAELB400 has been thoroughly investigated because it can
oxygenate a broad range of substrates. BphAELB400 variants
with extended substrate range have been generated by site-di-
rected mutagenesis (12) and directed evolution (13, 14). How-
ever, the enzyme’s structural features that modulate substrate
range and catalytic efficiency have yet to be determined. Many
investigations have identified residues in contact with the sub-
strate, or removed from it, as key determinants of substrate
preference and regiospecificity (12–23). Recently Thr335 of
BphAELB400, which is removed from the substrate, was found to
restrain the range of chlorobiphenyls the enzyme can oxidize by
controlling the spatial distribution of protein atoms in contact
with the substrates (17). Changing Thr335 to Ala relieves intra-
molecular constraints on Gly321, allowing for significant move-
ment of this residue during substrate binding, thereby increas-
ing the space available to accommodate the bulkier substrate
2,6-dichlorobiphenyl. In addition, crystal structures of the oxy-
genase component of carbazole 1,9a-dioxygenase and of the
binary complex of the oxygenase and ferredoxin components
provided evidence that conformational changes are required to
suitably align the Rieske clusters of the ferredoxin and oxyge-
nase components (24). These observations are consistent with a
mechanism whereby an induced fit process is involved in RO
substrate binding and catalytic function. Understanding how
conformational adjustments influence the turnover rate and
how they can be modified to enhance activity toward new sub-
strates will aid the development of new, better performing
catalysts.
Rieske-type oxygenases (ROs)3 catalyze a stereospecific oxy-
genation of many aromatic and hetero-aromatic molecules.
These enzymes have potential applications as biocatalysts to
degrade persistent pollutants, such as polyaromatic hydrocar-
bons (1, 2), polychlorinated biphenyls (3–5), and chlorodiben-
* This work was supported by Natural Sciences and Engineering Research
Council of Canada Grant RGPIN/39579-2007 and an Indian Department of
Science and Technology young scientist grant (to P. K.).
The atomic coordinates and structure factors (codes 2YFI and 2YFJ) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed: INRS-Institut Armand-
Frappier, Laval, Que´becH7V1B7, Canada. Tel.:450-687-5010;Fax:450-686-
5501; E-mail: Michel.Sylvestre@iaf.inrs.ca.
Unlike biphenyl, the fused rings of dibenzofuran are locked
in a co-planar conformation (Fig. 1), and this molecule is poorly
oxygenated by BphAELB400 (25, 26). In previous reports (7, 14),
3 The abbreviations used are: RO, Rieske-type oxygenase; IPTG, isopropyl ␤-D-
thiogalactopyranoside; MES, 4-morpholineethanesulfonic acid.
27612 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 31•AUGUST 5, 2011

Expanding RO Substrate Range
TABLE 1
Sequence pattern of BphAELB400 variants
Residue positiona
Protein designation
335
336
338
341
409
BphAELB400
T
A
T
A
A
A
A
A
A
A
A
F
N
N
N
N
Q
N
N
Q
N
Q
Q
I
L
L
L
L
L
L
F
L
F
F
F
F
I
M
M
M
M
M
M
M
M
M
I
BphAEp4
I
I
V
I
V
V
I
BphAERR41
V
a All other residues for these variants are identical to those of BphAELB400
.
DNA protocols were generally according to Sambrook et al.
(29). DNA from each mutant was sequenced at the Genome
Quebec DNA Sequencing Center (Montreal, Canada). Biphe-
nyl and dibenzofuran were of the highest purity grade available
from AccuStandard (New Haven, CT).
Protein Analysis—The level of expression of each variant
enzyme in IPTG-induced E. coli DH11S pDB31[LB400-bphFG] ϩ
pQE31[bphAE] was assessed by SDS-PAGE (30) of crude cell
extracts prepared under benign (cells were sonicated in 10 mM
phosphate buffer, pH 7.3, containing 140 mM NaCl) or denatur-
ating conditions (cells were sonicated in 10 mM phosphate
buffer, pH 7.3, containing 140 mM NaCl and 8 M urea). The gels
were stained with Coomassie Brilliant Blue. Purified enzyme
preparations were also analyzed by HPLC gel filtration chroma-
tography using a Waters Protein Pak 300 SW column (7.8 ϫ
300 mm), as described previously (31).
FIGURE 1. Biphenyl dioxygenase reaction. The inset shows the structure of
dibenzofuran and possible positions of oxygenation. A, angular; L, lateral.
Monitoring Enzyme Activity with Purified Enzyme Prep-
arations—Reconstituted His-tagged purified biphenyl dioxyge-
nase preparations were used to monitor enzyme activity and
metabolite production. In this case, the genes expressing each
enzyme component were cloned into pET-14b (Novagen, Mad-
ison, WI) and expressed in E. coli C41(DE3). The components
were produced as recombinant His-tagged protein and purified
by affinity chromatography on high performance nickel-Sep-
harose resin (GE Healthcare) (7). The concentration of each
purified component was determined by spectrophotometry
(31–33). Enzymatic reactions were performed as described pre-
viously (7) at 37 °C, in 50 mM pH 6.0 MES buffer, and in a
volume of 400 ␮l containing 1.2 nmol of each of the His-tagged
enzyme component and 200 nmol of NADH. Substrate deple-
tion and metabolite production were analyzed and quantified
by GC-MS using previously published protocols (7). The
steady-state kinetic parameters of all BphAEs were determined
we described variant BphAEp4 obtained by the double substitu-
tion of Thr335-Phe336 of BphAELB400 to Ala335-Met336 and var-
iant BphAERR41 obtained by changing Asn338, Ile341, and Leu409
of BphAp4 to Gln338, Val341, and Phe409. BphAERR41 was
selected by directed evolution for its higher turnover rate with
dibenzofuran than the parent BphAEp4 (7). The library was pro-
duced by changing Asn338 and Ile341 of BphAEp4 simultane-
ously by saturation mutagenesis (7), but an additional sponta-
neous mutation L409F occurred in this mutant.
In this study, to gain more insight into the pathways through
which ROs evolve to expand their substrate range, we identified
the mutations in BphAEp4 and in BphAERR41 that contribute
most to their enhanced activity toward dibenzofuran and ana-
lyzed the crystal structures of BphAERR41 and its dibenzofuran-
bound form to evaluate the consequences of the mutations.
EXPERIMENTAL PROCEDURES
Strains and Plasmids—Escherichia coli DH11S (27) and by recording oxygen consumption rates using a Clarke-type
C41(DE3) (28) (Statagene, La Jolla, CA) were used in this study. Hansatech model DW1 oxygraph (34) for concentrations of
The WT BphAELB400 and its mutants BphAEp401 (T335A), biphenyl and dibenzofuran varying between 5 and 150 ␮M.
BphAEp402 (F336M), BphAEp4 (T335A/F336M), and Kinetic parameters reported in this investigation were obtained
BphAERR41 (T335A/F336M/N338Q/I341V/L409F) were de- from analysis of at least two independently produced prepara-
scribed previously (17).
tions tested in triplicate.
A previously described two-step site-directed mutagenesis
Crystal Structure Analyses—Purification, crystallization, and
protocol (7) was used to create a set of mutants representing the preliminary x-ray diffraction properties of BphAERR41 have
six mutants that can be produced by single and double substi- been communicated elsewhere (35). The procedures to prepare
tutions of N338Q, I341V, and L309F of BphAEp4 (Table 1). crystals of BphAERR41 and its dibenzofuran-bound form were
These bphAE mutants were cloned in pQE31 or in pET14b. identical to those described for BphAEp4 (17). The crystal struc-
AUGUST 5, 2011•VOLUME 286•NUMBER 31
JOURNAL OF BIOLOGICAL CHEMISTRY 27613

Expanding RO Substrate Range
FIGURE 2. GC-MS spectra showing the relative amounts of the dihydrodihydroxy metabolites produced from dibenzofuran by BphAELB400 and its
variants. 1.2 nmol of each purified enzyme preparation was incubated for 2 min under the conditions described under “Experimental Procedures.” The
metabolites were extracted and derivatized with butylboronate before analysis by GC-MS.
TABLE 2
Steady-state kinetic parameters of BPDO variants
The steady-states kinetics were determined from the oxygen consumption rates as described under “Experimental Procedures.”
BpbAE
BphAERR41
Substrate
Biphenyl
BphAELB400 WT T335A
F336M
T335A/F3p346M
I341Va
I341V/L409Fa N338Q/L409Fa N338Q/I341V/L409Fa
Km (␮M)
22 (0.0)
0.9 (0.1)
41 (6.0)
30 (4.0)
17 (5.0)
33 (1.4)
1.0 (0.1)
31 (4)
32.5 (2.0) 21.5 (0.7)
32.0 (0.0)
1.5 (0.1)
46 (4.0)
34.5 (6.3)
1.3 (0.3)
38 (0.0)
Ϫ1
k
cat (s
)
1.1 (0.0) 0.4 (0.1)
0.7 (0.1)
21 (0.5)
0.8 (0.1)
37 (3.0)
Ϫ1
k
cat/Km (103
M
Ϫ1 s
)
)
36 (5.0)
23 (0.1)
Dibenzofuran
K
m (␮M)
19.5 (0.7)
0.12 (0.0)
6 (0.0)
19 (0.6)
19 (1)
20 (1.4)
25 (9.9)
21.5 (2.1)
23 (7.8)
0.5 (0.05)
22 (5)
22.0 (0.0)
0.38 (0.08)
17 (4.0)
Ϫ1
k
cat (s
)
0.22 (0.0) 0.12 (0.05) 0.26 (0.01)
0.17 (0.07) 0.22 (0.02)
Ϫ1
k
cat/Km (103
M
Ϫ1 s
12 (2.0)
6 (0.07)
13 (2.0)
7 (0.1)
10 (0.1)
a These mutants carry also the double T335A/F336M mutation.
tures were obtained and analyzed using the same approaches consumption rates recorded for variable concentrations of
and software used in studies of BphAEp4 (17). Crystal structures dibenzofuran (Table 2). These results show the superior ability
of BphAERR41 and its dibenzofuran-bound form were com-
pared with crystal structures of BphAELB400 (RCSB Protein
Data Bank accession code 2XR8) and its biphenyl-bound form
(code 2XRX), as well as BphAEp4 (code 2XSO) and its 2,6-di-
chlorobiphenyl-bound form (code 2XSH).
to metabolize dibenzofuran of BphAERR41 compared with
BphAEp4 and BphAELB400
.
To identify the mutations that contribute most to the
enhanced activity of BphAEp4 and BphAERR41 toward dibenzo-
furan, we assayed all mutants carrying single or multiple muta-
tions at positions 335, 336, 338, 341, and 409 (Table 1). Based on
the sum of areas under GC-MS peaks, the amounts of metabo-
lites produced by the T335A mutant and by BphAEp4 (T335A/
F336M) were similar and about three times higher than the
amounts for BphAELB400 and its F336M mutant (Fig. 2). The
superior ability of the T335A mutant to metabolize dibenzo-
furan was confirmed by steady-state kinetics (Table 2). Further-
more, the single F336M substitution lowered kcat and kcat/Km
for the reaction with biphenyl. This identified the T335A sub-
stitution as responsible for the enhanced ability of BphAEp4 to
metabolize dibenzofuran.
Protein Data Bank Accession Codes—The coordinates have
been deposited with the RCSB Protein Data Bank under acces-
sion codes 2YFI for BphAERR41 and 2YFJ for its complex with
dibenzofuran.
RESULTS
Steady-state Kinetics of BphAEp4, BphAERR41, and Their
Variants with Dibenzofuran—Based on the sum of areas under
GC-MS peaks of metabolites produced when 1.2 nmol of
enzyme was incubated for 2 min with 100 ␮M dibenzofuran,
BphAEp4 and BphAERR41 produced, respectively, three and
four times more metabolites than BphAELB400 (Fig. 2). Consis-
The apparent kcat values for the mutants T335A/F336M/
tent with the single time point measurements, the apparent kcat I341V and T335A/F336M/I341V/L409F toward biphenyl and
value for BphAERR41 is ϳ1.5 times higher than that of BphAEp4 dibenzofuran were lower than for BphAEp4. In addition, intro-
and 3 times higher than for BphAELB400 based on the oxygen ducing the single I341V or the double I341V/L409F mutations
27614 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 31•AUGUST 5, 2011

Expanding RO Substrate Range
into BphAEp4 did not contribute to enhanced activity toward
dibenzofuran. We did not determine steady-state kinetic
parameters for variant T335A/F336M/L409F. However, the
observation that recombinant E. coli cells producing this
enzyme did not degrade dibenzofuran more efficiently than
BphAEp4 (not shown) indicates that changing Leu409 of
BphAEp4 to Phe did not influence activity toward dibenzofuran.
Variants T335A/F336M/N338Q and T335A/F336M/N338Q/
I341V were poorly active toward biphenyl and dibenzofuran
because they are not assembled correctly (see below). The
steady-state kinetic parameters obtained with these two vari-
ants were too difficult to determine accurately and therefore are
not reported here. On the other hand, the apparent kcat value
for T335A/F336M/N338Q/L409F toward dibenzofuran was in
the same range or even higher than for BphAERR41 (T335A/
F336M/N338Q/I341V/L409F) (Table 2). Furthermore, replac-
ing Asn338 and Leu409 of BphAEp4 with Gln338 and Phe409 cre-
ated a double mutant exhibiting enhanced activity toward
dibenzofuran.
The Poor Activity of Variants T335A/F336M/N338Q and
T335A/F336M/N338Q/I341V Is the Result of Hexamer
Misassembly—Unlike BphAERR41 and T335A/F336M/N338Q/
L409F, variants T335A/F336M/N338Q and T335A/F336M/
N338Q/I341V were poorly active, which suggests a detrimental
effect of the N338Q substitution on enzyme activity. When the
proteins of IPTG-induced recombinant E. coli cells producing
variant BphAEs were extracted under denaturing conditions
and separated by SDS-PAGE, the intensities of bands corre-
sponding to the ␣ and ␤ subunits were similar for all strains
(Fig. 3A). This shows that the level of expression of BphAE is
similar for all IPTG-induced E. coli clones expressing these
variants. However, analysis of cell extracts prepared under non-
denaturing conditions revealed significantly lower intensities of
the bands corresponding to the ␣ subunits for variant T335A/
F336M/N338Q and T335A/F336M/N338Q/I341V compared
with the other variants (Fig. 3B). This suggests misassembly or
early dissociation of ␣3␤3 hexamers resulting in a loss of ␣ sub-
units into the nonsoluble protein fraction. HPLC gel filtration
analysis of freshly purified His-tagged BphAE of variants
T335A/F336M/N338Q and T335A/F336M/N338Q/I341V
confirmed that less than 10% of each protein preparation exhib-
ited the expected ␣3␤3 association pattern (not shown). There-
fore, the N338Q substitution hinders hexamer assembly unless
a concomitant L409F substitution is introduced. Nevertheless,
the double N338Q/L409F substitution is beneficial for enhanc-
ing catalytic properties toward dibenzofuran.
FIGURE3.SDS-PAGEoflyzatesofIPTG-inducedE. colicellsexpressingthe
indicated variants derived from BphAEp4 (T335A/F336M). A, gel of the
indicated protein extracts prepared under denaturing conditions. B, gel of
the indicated protein extracts prepared under nondenaturing conditions. In
addition to the indicated substitutions, all of the mutants in the fourth lane
through the eighth lane carry the double T336A/F336M mutation. The far-left
lane is the molecular weight marker. The bands corresponding to the ␣ and ␤
subunits are marked by arrows.
poorly ordered residues and protein segments were the same as
Overall Structure of BphAERR41—The overall structures of observed for BphAEp4 (17), including segments comprising res-
BphAERR41 and its dibenzofuran-bound form are very similar idues Ile247 to Lys263 and Glu280 to Val287 of the ␣ subunit and
to the crystal structures of the BphAEp4:2,6-dichlorobiphenyl residues 9–17 and 158–164 of the ␤ subunit. In the
complex (17), which contains triplets of ␣␤ dimers associated BphAERR41-dibenzofuran complex structure, dibenzofuran
into two hexamers in the asymmetric unit (chains ABCDEF and could be identified clearly in Fo Ϫ Fc difference Fourier maps in
chains GHIJKL). For both structures, the final refined models all active sites of the ABCDEF hexamer. Similar to BphAELB400
-
contain residues Asn18 to Phe143 plus Phe153 to Pro459 of each ␣ biphenyl, a water molecule lies between the Fe2ϩ and dibenzo-
subunit, residues Phe9 to Phe188 of each ␤ subunit and 1217 furan at ϳ2 Å from the catalytic iron. Electron density maps of
water molecules. The diffraction data and refined models are the active site residues of the BphAERR41 and of its dibenzo-
characterized in Table 3.
furan-bound form are shown for dimer AB in Fig. 4.
␣
Superposition of all C atoms for the ␣␤ dimer of chains AB
When the BphAERR41-dibenzofuran BphAELB400-biphenyl
with chains CD-KL yielded rmsd values of 0.2–0.3 Å. The structures are superposed, the positions of the carbons targeted
AUGUST 5, 2011•VOLUME 286•NUMBER 31
JOURNAL OF BIOLOGICAL CHEMISTRY 27615

Expanding RO Substrate Range
for hydroxylation, C-4a and C-4 of dibenzofuran and C-2 and crystal structure, the furan ring’s oxygen atom contacts the
water ligand of the active site Fe2ϩ atom. Based on observations
drawn from the naphthalene dioxygenase crystal structure,
Karlsson et al. (36) proposed a reaction cycle for ROs in which
Fe2ϩ coordinates this water in states prior to dioxygen binding.
When dioxygen binds, it intercalates side-on between the iron
and the substrate displacing the water. Thus, a subsequent
adjustment in substrate position or orientation is possible such
that the lateral attack would occur. In essence, the dibenzo-
furan-O-water interaction seen in the crystal form could pro-
duce a false inference about the points of attack because the
structure reports on a state prior to dioxygen binding.
C-3 of biphenyl, are nearly the same in ␣␤ dimers AB, CD, and
EF (not shown). In ␣␤ dimers AB, CD, and EF of BphAERR41
-
dibenzofuran, the substrate is in an orientation that would favor
4,4a angular attack. This is unexpected because biochemical
data revealed dioxygenation of the lateral 1,2 and 3,4 carbons of
dibenzofuran as by far most favored for BphAERR41 (7). In the
TABLE 3
Crystallographic data and refinement results for BphAERR41 structure
BphAERR41
BphAERR41:dibenzofuran
Crystallographic data
Space group
Wavelength
Resolution
Cell dimensions
a (Å)
P21
P21
Structural Analysis of the Influence of Residue 335 on Cata-
lytic Properties toward Dibenzofuran—The average active site
cavity volume of the ␣␤ dimers AB, CD, and EF of BphAERR41
was comparable with that of BphAEp4 (17) (1073 Å3 as calcu-
lated using CASTp program (37)). The corresponding atoms of
the reactive ring of dibenzofuran and of biphenyl interact with
the same residues of the ␣ subunits of BphAERR41 and
0.9
0.9
100-2.2
100-2.2
86.9
86.9
b (Å)
277.8
92.9
278.1
c (Å)
92.9
␣ (°)
90.0
90.0
␤ (°)
117.6
90.0
117.6
␥ (°)
90.0
Unique reflections
208106
210175
92.9 (80.3)
8.0 (55.0)
16.0 (2.0)
4.4 (2.5)
BphAELB400 (Gln226, Phe227, Asp230, Met231, Leu233, Ala234
,
Completeness (%) (last shell) 99.0 (94.0)
R
sym (%) (last shell)a
7.0(2)
His323, and Leu333), and they are located at approximately the
same distances (not shown). Therefore, neither the overall size
of the cavity nor the constraints on the reactive ring are affected
by the mutations introduced in BphAERR41. The residues lining
the distal portion of the BphAERR41 catalytic cavity are the same
I/␴ (last shell)
17.4 (2.2)
3.7 (3.0)
Multiplicity (last shell)
Refined model
No. of residues
3720
1217
100–2.2
17.6
3720
1287
100–2.2
19.7
Water molecules
Resolution range (Å)
R
fact (%)
free (%)
as those in BphAELB400 and BphAEp4 (Phe384, Phe378, Val287
,
R
22.2
22.9
Average B-factors (Å2)
Ser283, Phe/Met336, Leu333, Gly321, Tyr277, His239, Ala234, and
Met231) (Fig. 5). However, the overall shape of the cavity of
BphAERR41-dibenzofuran differs significantly from that of
BphAELB400-biphenyl but is similar to that of BphAEp4:2-chlo-
robiphenyl (Fig. 5). This is caused principally by the replace-
ment of Phe336 by Met combined with the conformational free-
dom of Gly321. The new catalytic properties of BphAEp4 toward
dibenzofuran (and in part those of BphAERR41) can thus be
attributed to structural changes in the distal portion of the sub-
strate-binding pocket. As noted for BphAEp4, the T335A muta-
tion relieves constraints on the Val320:Gln322 segment, allowing
Protein chains
AB 42.4, 43.2 AB 42.3, 42.5
CD 42.6, 43.3 CD 42.2, 42.7
EF 44.7, 44.0
EF 43.4, 43.2
GH 47.1, 45.5 GH 42.2, 42.9
IJ 47.7, 46.9 IJ 42.9, 42.4
KL 48.4, 45.9 KL 42.9, 41.6
Waters
49.4
42.6
All atoms
30803
0.01
30992
0.01
Bond lengths (Å)
Bond angles (°)
Ramachandran plot (%)
Preferred
1.33
0.91
89.3
10.6
0.1
n
89.3
10.6
0.1
Allowed
Outliers
a Rsym ϭ ⌺hkl
⌺
͉Ihkl,i Ϫ Ihkl͉/⌺hkl
⌺
iϭ1Ihkl,i
.
n
៮
iϭ1
FIGURE 4. The 2Fobs ؊ Fcalc electron density contoured at 1.0 ␴ level in the vicinity of the active site of chain AB from BphAERR41. A, substrate-free.
B, dibenzofuran-bound form.
27616 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 31•AUGUST 5, 2011

Expanding RO Substrate Range
FIGURE 5. Superposition of a portion of the catalytic cavity showing the residues lining the distal ring pocket. Superposition of dimers AB of BphAERR41
dibenzofuran (yellow) and BphAELB400-biphenyl (red) (A) and BphAERR41-dibenzofuran (yellow) and BphAEp4:2,6-dichlorobiphenyl (green) (B).
-
displacement of the Gly321 carbonyl such that it moves away contacts a short segment (␤21) comprised of residues Pro408
,
from the substrate (17). In this case, the removal of Gly321 from Phe409, and Asn410 and located at the edge of the catalytic
dibenzofuran reduces the influence it exerts on the distal ring of domain. Agr103 forms a polar contact with Glu225 of helix ␣6.
the substrate. This is significant because dibenzofuran is oblig- Arg101 forms polar contacts with Pro408 and Asn410, and Arg104
atory co-planar: an altered placement of the distal ring would forms a polar contact with Asn410 (Fig. 6C). Therefore, the crys-
influence the orientation of the proximal ring inside the cata- tal structures show that residue Phe409 is located within a
lytic pocket.
stretch of amino acids that appears to play an important role in
Structural Analysis of the Influence of Residues 338 and 409 subunit assembly and/or in maintaining the stability of the oli-
on Enzyme Stability—Based on the crystal structure of gomeric structure. In BphAERR41, Phe409 is ϳ4.6 Å from Phe222
BphAERR41, as well as the structures of BphAELB400 and of helix ␣6 (Fig. 6C). Alignment of dimers AB, CD, EF, GH, IJ,
BphAEp4 (17), Gln338 and Phe409 are too distant from each and KL of BphAERR41 and its dibenzofuran-bound form with
other to interact. To understand the effect of these two muta- dimers AB, CD, EF, GH, IJ, and KL of the substrate-free and
tions,we need to examine closely the overall structure of the ␣ bound forms of BphAELB400 or BphAEp4 shows that Phe409 of
and ␤ subunits and the contacts at the ␣␣, ␤␤, and ␣␤ BphAERR41 aligns very well with Leu409 of BphAELB400 or
interfaces.
BphAEp4 (not shown). In the latter enzymes, however, Leu409 is
The overall crystal structure of the ␤ subunit of BphAERR41 is at an average distance of 5.4 Å from Phe222. This could explain
very similar to that of other biphenyl dioxygenases (16, 17) and why replacing Leu409 of BphAEp4 with a larger side chain in
naphthalene dioxygenase (2). It includes a long twisted six- Phe409 suppresses the negative impact of the N338Q mutation
stranded ␤ sheet, with three helices on the inward side of the on hexamer assembly. Through its interaction with Phe222
,
sheet and a loop made of residues 9–20 (Fig. 6A) on its outward Phe409 seems to help stabilize subunit assembly by reinforcing
side. Many polar interactions are uniformly distributed the role played by segment Pro408–Asn410 in holding the sub-
between the ␤-sheet residues of vicinal ␤ subunits and between units together.
two of the helices and ␤-sheet residues of the vicinal subunit. In
Structural Analysis of the Influence of Residues 338 and 409
addition, helix ␣3 and strand ␤2 are in contact with the ␣ sub- on Catalytic Properties—Prior studies identified mutations
unit. This suggests that the ␤ subunit plays a key role in subunit within a subsequence called region III that influenced the oxy-
assembly.
genase’s catalytic properties (12–14). This region includes a
As reported for other dioxygenases (2, 16), the ␣ subunit loop between strands ␤18 and ␤19 and a portion of strand ␤19.
comprises two domains: the Rieske and catalytic domains. Residues 338 and 341 are both located on strand ␤19 (Fig. 6C).
Unlike the ␤ subunits, the crystal structures show unevenly Superposition of the catalytic domains of BphAERR41 and
distributed contacts between vicinal ␣ subunits; these contacts BphAELB400 reveals minor variations in strand ␤19 that can be
occur principally at the junction between the Rieske domain of attributed to the longer side chain of Gln338 in BphAERR41 (not
one ␣ subunit and the catalytic domain of its vicinal subunit shown). Strand ␤19 faces helix ␣12, which interacts with the tip
(Fig. 6A).
of hairpin-2 of the vicinal Rieske domain and includes iron
The Rieske domain is dominated by antiparallel ␤ strands ligand Asp388. The tip of Rieske domain hairpin-2 includes
from which two hairpin structures protrude to form two fingers Ser121, Tyr122, and His123. These residues make polar contacts
that hold the [2Fe—2S] center. The catalytic domain contains with Thr237 and Thr238 located at the junction between helices
the catalytic Fe2ϩ, which lies against a eight-stranded antipar- ␣7 and ␣8, on which are located iron ligands His233 and His239
.
allel ␤ sheet on one side and is surrounded on the other sides by In addition, Ser121 and His123 make polar contacts with Gln226
helices and loops (Fig. 6B). Residues Arg101, His102, Arg103, and and Asp230, two residues believed to be involved in the reaction
Gly104 of the Rieske domain form the tip of hairpin 1. This short mechanism (36, 38) and found in the catalytic cavity at the level
segment (in blue in Fig. 6, B and C) is embedded inside a match- of the proximal ring of the substrate (17) (Fig. 6B). Further-
ing trough of the vicinal ␣ subunit; it faces helix ␣6 of the cata- more, Tyr122 has a polar contact with Trp392 and Ser121 with
lytic domain, comprised of residues Trp220 to Ser229, and it also Asn391 of helix ␣12. Gln338 forms two polar contacts with
AUGUST 5, 2011•VOLUME 286•NUMBER 31
JOURNAL OF BIOLOGICAL CHEMISTRY 27617

Expanding RO Substrate Range
27618 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 31•AUGUST 5, 2011

Expanding RO Substrate Range
Arg340, and the latter forms two polar contacts with Glu385 of
helix ␣12. Arg340 is also close enough to Glu385 to form a salt
bridge with this residue (Fig. 6C). Therefore, Arg340 and Gln338
are located such that their conformation can influence the dis-
tribution in the space of helices ␣12, ␣8, ␣7, and ␣6, which are
critical for catalytic activity and subunit assembly.
Analysis of the crystal structure did not suggest a clear-cut
mechanism by which Gln338 and Arg340 exert these effects.
However, the longer length of the Gln338 side chain compared
with Asn338 might disturb a key state not observed in the crystal
or the internal dynamics of the protein. With respect to the
latter possibility, it is clear from structural analysis that residues
on helix ␣12 move considerably during substrate binding,
showing that this protein segment is rather adaptable (Fig. 7).
This movement is likely required to suitably align the reactive
atoms for progression along the chemical reaction.
In ROs, the proximity between the Rieske cluster and the iron
in the active site of the adjacent ␣ subunit is consistent with a
mechanism involving a transfer of electron across the interface
between two subunits (2, 16, 17). This is corroborated by the
fact that full activity requires that ␣ and ␤ subunits associate
into a ␣3␤3 configuration (31). Such a mechanism must
demand precise alignment of the amino acids involved in elec-
tron transfer between the Rieske cluster and the mononuclear
iron of each adjacent ␣ subunit, highlighting the importance of
the protein atoms involved in the ␣␣ subunit interface.
Therefore, crystal structure analysis is consistent with an
induced fit response required to reorganize the active site and
facilitate the interplay between protein atoms critical for the
reaction. The N338Q substitution might disturb the conforma-
tion of helices ␣12 and ␣6, resulting in subunit instability or
misassembly. Conversely, the double Gln338 and Phe409 muta-
tion may affect the conformational fluctuations of these helices
in such a way that it enhances the roles of protein residues such
as Asn388, Gln226, and Asp230 that are located on the helices and
presumed to be involved in the chemical steps of the reaction
(2, 38).
DISCUSSION
In this study, we examined the crystal structure of
BphAERR41, an evolved RO that oxidizes dibenzofuran more
efficiently than its BphAELB400 and BphAEp4 parents. Despite
the limitations of crystal structure analyses, the study revealed
two pathways through which ROs evolve to expand their sub-
strate range.
FIGURE 7. Superposition of residues of the catalytic domain that move
after substrate binding. A, residues of BphAERR41 (red) and BphAERR41
-
dibenzofuran (green). B, residues of BphAELB400 (brown) and BphAELB400-bi-
phenyl (yellow). C, residues of BphAEp4 (gray) and BphAEp4:2,6-dichlorobiphe-
nyl (purple).
Traditionally, enzyme engineering to alter the substrate
range involves mutations at residues lining the catalytic pocket.
This approach has been applied successfully in many circum- catalytic properties of biphenyl dioxygenase toward biphenyl
stances (39–45). Reducing the size of a side chain or altering analogs (21, 22, 44). In this work we confirm the importance of
charge distributions can generate enzyme with new catalytic the T335A mutation. In altering the plasticity of the catalytic
properties.
cavity, this mutation allows the carbonyl of residue Gly321 to
However, other studies have shown that several residues not move away from the substrate. In a previous work, we showed
in direct contact with the substrate can significantly change the that this movement was required to increase the space available
FIGURE 6. A, backbone ribbon drawing of the vicinal ␣/␤ dimers AB (green and salmon) and CD (red and purple) of BphAERR41-dibenzofuran highlighting the
numerous contacts between two vicinal ␤ subunits. B, ribbon drawing of the catalytic domain of monomer C (red) and of the Rieske domain of monomer A
(green) of BphAERR41-dibenzofuran highlighting the interface between the two monomers. C, close-up view of the same interface shown in B. Hairpins 1 and 2
from the Rieske domains of monomer A (in blue and gray) protrude into a matching trough of the vicinal catalytic domain of monomer C. The residues from
monomer C that contact the hairpins from the vicinal subunit are colored in blue or gray to match the residues they contact.
AUGUST 5, 2011•VOLUME 286•NUMBER 31
JOURNAL OF BIOLOGICAL CHEMISTRY 27619

Expanding RO Substrate Range
to bind the bulky 2,6-dichlorobiphenyl in a productive orienta- in the catalytic reaction (36), moved significantly during sub-
tion (17). Because dibenzofuran is obligatory co-planar, any strate binding in all variants (BphAELB400, BphAEp4, and
misplacement of the distal ring would influence the orientation BphAERR41).
of the proximal ring inside the catalytic pocket. Therefore, con-
Altogether, our analysis shows that evolving ROs to change
sistent with an induced fit mechanism, in BphAEp4 and their substrate specificity is a rather complex enterprise that
BphAERR41, the displacement of Gly321 appears to be required does not exclusively involve mutations at key residues in direct
to reduce the influence it exerts through atomic interactions on contact with the substrate. It appears that some mutations
the substrate’s distal ring.
affect key residues associated with necessary conformational
In this work, we highlighted a second and more subtle route changes that are more difficult to identify by a rational
to changes in substrate range, which implies that in ROs, either approach but that are required to allow productive or improved
one or both of the induced fit or protein dynamic processes are interplay of reaction-critical atoms both inside and outside the
involved to place the protein atoms involved in the reaction into substrate-binding pocket.
proper relationships that facilitate catalysis. The reaction cata-
lyzed by ROs is complex; it not only involves substrate binding
and release of product, but also one dioxygen molecule is
required in the reaction, and electrons must be transferred
from the ferredoxin component to the Rieske cluster of one ␣
subunit and then to the catalytic iron of the vicinal ␣ subunit.
Furthermore, a recent report showed residues at the interface
between the Rieske domain and the catalytic domain move dur-
ing formation of the complex between the oxygenase and ferre-
doxin components of carbazole 1,9a-dioxygenase (24). This
implies that reaction-critical atoms from the Rieske domain
must align properly with those of the vicinal catalytic domain,
and the reaction-critical atoms of the catalytic domain must
align properly to work together during the catalytic process.
Structural analysis shows that residues located on secondary
structures ␣6 and ␣12 are involved in subunit assembly, and
biochemical data suggest that they are involved in the catalytic
reaction (electron transfer and protonation) (2, 38). The fact
that these residues move during substrate binding is consistent
with a substrate-induced retuning process required to suitably
align the protein atoms involved in the chemical steps of the
reaction. In such a context, by altering the interactions occur-
ring between secondary structure elements surrounding the
catalytic center, the N338Q mutation generates a protein
unable to stabilize the ␣3␤3 assembly previously shown to be
required for activity (31). However, the double N338Q and
L409F substitution generates an ␣ subunit that supports a sta-
ble hexamer and where the retuning process is improved com-
pared with its BphAELB400 and BphAEp4 parents, resulting in a
more efficient and faster catalytic reaction. ROs can thus be
engineered to enhance their catalytic properties toward new
substrates by altering the process involved in fine-tuning the
interplay between the reaction-critical atoms.
Acknowledgment—We appreciate the use of the SE Regional Collab-
orative Access Team 22-ID Beamline in the collection of x-ray diffrac-
tion data.
REFERENCES
1. Jakoncic, J., Jouanneau, Y., Meyer, C., and Stojanoff, V. (2007) Biochem.
Biophys. Res. Commun. 352, 861–866
2. Kauppi, B., Lee, K., Carredano, E., Parales, R. E., Gibson, D. T., Eklund, H.,
and Ramaswamy, S. (1998) Structure 6, 571–586
3. Furukawa, K., Suenaga, H., and Goto, M. (2004) J. Bacteriol. 186,
5189–5196
4. Haddock, J. D., Horton, J. R., and Gibson, D. T. (1995) J. Bacteriol. 177,
20–26
5. Sylvestre, M. (2004) Int. Biodeter. Biodegr. 54, 153–162
6. Chang, Y. S. (2008) J. Mol. Microbiol. Biotechnol. 15, 152–171
7. Mohammadi, M., and Sylvestre, M. (2005) Chem. Biol. 12, 835–846
8. Chun, H. K., Ohnishi, Y., Shindo, K., Misawa, N., Furukawa, K., and Horin-
ouchi, S. (2003) J. Mol. Catal. B Enzym. 21, 113–121
9. Seeger, M., Gonza´lez, M., Ca´mara, B., Mun˜oz, L., Ponce, E., Mejías, L.,
Mascayano, C., Va´squez, Y., and Sepu´lveda-Boza, S. (2003) Appl. Environ.
Microbiol. 69, 5045–5050
10. Seo, J., Kang, S. I., Won, D., Kim, M., Ryu, J. Y., Kang, S. W., Um, B. H., Pan,
C. H., Ahn, J. H., Chong, Y., Kanaly, R. A., Han, J., and Hur, H. G. (2011)
Appl. Microbiol. Biotechnol. 89, 1773–1782
11. Erickson, B. D., and Mondello, F. J. (1992) J. Bacteriol. 174, 2903–2912
12. Mondello, F. J., Turcich, M. P., Lobos, J. H., and Erickson, B. D. (1997)
Appl. Environ. Microbiol. 63, 3096–3103
13. Barriault, D., Plante, M. M., and Sylvestre, M. (2002) J. Bacteriol. 184,
3794–3800
14. Barriault, D., and Sylvestre, M. (2004) J. Biol. Chem. 279, 47480–47488
15. Ferraro, D. J., Okerlund, A. L., Mowers, J. C., and Ramaswamy, S. (2006) J.
Bacteriol. 188, 6986–6994
16. Furusawa, Y., Nagarajan, V., Tanokura, M., Masai, E., Fukuda, M., and
Senda, T. (2004) J. Mol. Biol. 342, 1041–1052
17. Kumar, P., Mohammadi, M., Viger, J. F., Barriault, D., Gomez-Gil, L.,
Eltis, L. D., Bolin, J. T., and Sylvestre, M. (2011) J. Mol. Biol. 405,
531–547
Many questions remain unanswered; crystal structure anal-
ysis did not determine a clear-cut mechanism by which the
double N338Q/L409F substitution affects the enzyme structure
and catalytic properties, and our data do not determine which
of the enzymatic steps are accelerated during the reaction.
Although the data do not provide any direct demonstration that
the N338Q and L409F substitutions either affect an induced fit
or protein dynamic mechanism involved in the catalytic reac-
tion, it is clear from crystal structure analysis that these residues
occupy strategic positions whereby they can interact with reac-
tion-critical protein atoms/groups and affect oligomeric
assembly. Furthermore, residues of helix ␣12 and ␣6, and espe-
cially Asp388 and Gln226, which are postulated to play a key role
18. Suenaga, H., Goto, M., and Furukawa, K. (2001) J. Biol. Chem. 276,
22500–22506
19. Suenaga, H., Mitsuoka, M., Ura, Y., Watanabe, T., and Furukawa, K. (2001)
J. Bacteriol. 183, 5441–5444
20. Suenaga, H., Nishi, A., Watanabe, T., Sakai, M., and Furukawa, K. (1999)
J. Biosci. Bioeng. 87, 430–435
21. Ve´zina, J., Barriault, D., and Sylvestre, M. (2007) J. Bacteriol. 189, 779–788
22. Zielinski, M., Kahl, S., Hecht, H. J., and Hofer, B. (2003) J. Bacteriol. 185,
6976–6980
23. Zielinski, M., Kahl, S., Standfuss-Gabisch, C., Ca´mara, B., Seeger, M., and
Hofer, B. (2006) Appl. Environ. Microbiol. 72, 2191–2199
24. Inoue, K., Ashikawa, Y., Umeda, T., Abo, M., Katsuki, J., Usami, Y., Nogu-
chi, H., Fujimoto, Z., Terada, T., Yamane, H., and Nojiri, H. (2009) J. Mol.
Biol. 392, 436–451
27620 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 31•AUGUST 5, 2011

Expanding RO Substrate Range
25. L’Abbe´e, J. B., Barriault, D., and Sylvestre, M. (2005) Appl. Microbiol. Bio- 36. Karlsson, A., Parales, J. V., Parales, R. E., Gibson, D. T., Eklund, H., and
technol. 67, 506–514
Ramaswamy, S. (2003) Science 299, 1039–1042
26. Seeger, M., Ca´mara, B., and Hofer, B. (2001) J. Bacteriol. 183, 3548–3555 37. Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y., and Liang, J.
27. Lin, J. J., Smith, M., Jessee, J., and Bloom, F. (1992) BioTechniques 12,
718–721
(2006) Nucleic Acids Res. 34, W116–W118
38. Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181,
1831–1837
28. Miroux, B., and Walker, J. E. (1996) J. Mol. Biol. 260, 289–298
29. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A 39. Bak-Jensen, K. S., Andre´, G., Gottschalk, T. E., Pae¨s, G., Tran, V., and
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY
Svensson, B. (2004) J. Biol. Chem. 279, 10093–10102
40. Dietrich, M., Do, T. A., Schmid, R. D., Pleiss, J., and Urlacher, V. B. (2009)
J. Biotechnol. 139, 115–117
30. Laemmli, U. K. (1970) Nature 227, 680–685
31. Hurtubise, Y., Barriault, D., and Sylvestre, M. (1996) J. Biol. Chem. 271, 41. Huang, W. C., Westlake, A. C., Mare´chal, J. D., Joyce, M. G., Moody, P. C.,
8152–8156
and Roberts, G. C. (2007) J. Mol. Biol. 373, 633–651
42. Ostrander, E. L., Larson, J. D., Schuermann, J. P., and Tanner, J. J. (2009)
Biochemistry 48, 951–959
32. Haddock, J. D., and Gibson, D. T. (1995) J. Bacteriol. 177, 5834–5839
33. Hurtubise, Y., Barriault, D., Powlowski, J., and Sylvestre, M. (1995) J. Bac-
teriol. 177, 6610–6618
43. Parales, R. E., Lee, K., Resnick, S. M., Jiang, H., Lessner, D. J., and Gibson,
D. T. (2000) J. Bacteriol. 182, 1641–1649
34. Imbeault, N. Y., Powlowski, J. B., Colbert, C. L., Bolin, J. T., and Eltis, L. D.
(2000) J. Biol. Chem. 275, 12430–12437
44. Suenaga, H., Goto, M., and Furukawa, K. (2006) Appl. Microbiol. Biotech-
nol. 71, 168–176
35. Kumar, P., Go´mez-Gil, L., Mohammadi, M., Sylvestre, M., Eltis, L. D., and
Bolin, J. T. (2011) Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 67, 45. Xu, D., Enroth, C., Lindqvist, Y., Ballou, D. P., and Massey, V. (2002)
59–62
Biochemistry 41, 13627–13636
AUGUST 5, 2011•VOLUME 286•NUMBER 31
JOURNAL OF BIOLOGICAL CHEMISTRY 27621

Enzymology:
Retuning Rieske-type Oxygenases to
Expand Substrate Range
Mahmood Mohammadi, Jean-François Viger,
Pravindra Kumar, Diane Barriault, Jeffrey T.
Bolin and Michel Sylvestre
J. Biol. Chem. 2011, 286:27612-27621.
doi: 10.1074/jbc.M111.255174 originally published online June 8, 2011
Access the most updated version of this article at doi: 10.1074/jbc.M111.255174
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
This article cites 44 references, 23 of which can be accessed free at
http://www.jbc.org/content/286/31/27612.full.html#ref-list-1