Targeted Mutations in a Trametes villosa Laccase
AXIAL PERTURBATIONS OF THE T1 COPPER*
Feng
Xu
§,
Amy E.
Palmer¶,
Debbie S.
Yaver
,
Randy M.
Berka
,
Gregory A.
Gambetta
,
Stephen H.
Brown
, and
Edward
I.
Solomon¶
From
Novo Nordisk Biotech, Davis, California 95616 and the ¶ Department of Chemistry, Stanford University,
Stanford, California 94305
 |
ABSTRACT |
Trametes villosa laccase was mutated
on a tetrapeptide segment near the type 1 site. The mutations F463M and
F463L were at the position corresponding to the type 1 copper axial
methionine (M517) ligand in Zucchini ascorbate oxidase. The
mutations E460S and A461E were near the T1 copper site. The mutated
Trametes laccases were expressed in an Aspergillus
oryzae host and characterized. The E460S mutation failed to
produce a transformant with meaningful expression. The F463L and A461E
mutations did not significantly alter the molecular and enzymological
properties of the laccase. In contrast, the F463M mutation resulted in
a type 1 copper site with an EPR signal intermediate between that of
the wild type laccase and plastocyanin, an altered UV-visible spectrum,
and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an
increase in kcat and Km.
These effects are attributed to a significant perturbation of the T1
copper center caused by the coordination of the axial methionine (M463) ligand.
 |
INTRODUCTION |
Laccase (p-diphenol:dioxygen oxidoreductase, EC
1.10.3.2) is a copper-containing oxidase that couples the oxidation of
substrate (usually diphenols, aryl diamines, or amino phenols) with the reduction of dioxygen to water (1-4). Sequence comparisons, crystal structure analyses, and spectroscopy indicate that all multicopper oxidases contain at least one type 1 (T1)1 copper center, one type
2 (T2) copper center, and one type 3 (T3) copper center (3-7). These
copper sites are defined by their spectroscopic properties. The T1, or
"blue," copper is characterized by an intense (
~ 5000 M
1 cm
1) absorption band around
600 nm and an unusually small (<100 × 10
4
cm
1) parallel hyperfine coupling in EPR. The T2, or
normal, copper site does not exhibit a strong feature in the visible
absorption spectrum, but has a parallel hyperfine coupling > 160 × 10
4 cm
1. The T3, or coupled
binuclear, copper center consists of two copper atoms connected by a
hydroxide bridge. This bridge provides a strong superexchange pathway
and therefore mediates antiferromagnetic coupling. The T3 center is
EPR-silent, but is characterized by an absorption band around 330 nm
(
~ 5000 M
1 cm
1). The T2
and T3 sites form a trinuclear copper cluster that is the site for
O2 reduction (5, 8, 9). Laccase is the simplest of the
multicopper oxidases, containing one of each type of copper site for a
total of 4 copper atoms.
Based on a wide range of comparative studies, including sequence
homology and crystal structure analysis, the copper site coordination
is very similar among the multicopper oxidases. The main difference is
in the coordination sphere of the T1 copper site. The typical T1 site,
such as that found in the blue copper protein plastocyanin (Pc) and the
multi-copper protein Zucchini ascorbate oxidase (zAO),
contains two histidines (His), a cysteine (Cys) that forms a short S-Cu
bond, and a methionine (Met) that forms a long S-Cu bond. These four
ligands bind T1 copper in a distorted tetrahedral coordination geometry
(Fig. 1, A and B). In contrast, most fungal
laccases with known primary sequence have either a leucine (Leu) or
phenylalanine (Phe) at the position corresponding to the axial Met
ligand (3, 5). Neither Leu nor Phe would be expected to coordinate to
the copper that would render a tri-coordinate T1 site. The recent
crystal structure of Coprinus cinereus laccase (CcL)
confirms this and shows that the T1 site has only three ligands (two
His and one Cys) in a trigonal planar geometry (Fig.
1C) (7). An interesting
question is how this difference in geometry is manifested in terms of
differences in the electronic structure and the electron transfer
function of the T1 site. The redox potential (E°) of
fungal laccases ranges from 0.48 V to 0.78 V, whereas Pc and zAO have
E° in the range of 0.3 to 0.4 V (3). It has been proposed
that the lack of an axial ligand might be responsible for the high
E° observed in some fungal laccases (10-12), as it is
expected that the elimination of the axial Met donor interaction would
preferentially stabilize the reduced Cu(I) state. A number of
mutagenesis studies (13-15) have been performed on the T1 copper site
in azurin, and it was found that the nature of the axial ligand can
influence E° to some extent. Mutation of Met-121 was found
to tune the E° over a range of
0.105 V to 0.138 V with
respect to the E° of the wild-type (wt) azurin at pH
7.0.

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Fig. 1.
Comparison of the ligation of the T1 copper
in Pc (Protein Data Bank code 1PLC) (A), zAO (Protein
Data Bank code 1AOZ) (B), and CcL (Protein Data Bank
code 1A65) (C). In Pc, the Cu-SM92
bond length is 2.82 Å; in zAO, the Cu-SM517 bond length is
2.90 Å; in CcL, the distance between the copper and L462 is ~3.5
Å.
|
|
The present study utilized site-directed mutagenesis to examine how the
composition of the amino acid residues in the vicinity of the T1 copper
affect the structure and E° of this copper in a
Trametes villosa laccase (TvL) (16). We were also interested in determining how the mutations would impact the enzymatic properties, kcat, Km, and the pH
dependence of enzymatic activity. TvL has a primary structure and
redox/enzymatic properties very similar to the Trametes
(Polyporus or Coriolus) versicolor laccase (PvL), an
enzyme that has been extensively studied and is regarded as a
representative high E° laccase (1-4). We mutated Phe-463, which corresponds to the axial Met-517 in zAO, and two other residues (Glu-460 and Ala-461), corresponding to the His-514 and Met-515 in zAO
(Table I). Our results showed that the
F463M mutation led to significant changes in the T1 copper site,
decreased the E°, and altered kcat
and Km.
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Table I
Sequence alignment between zAO, TvL, and CcL
Underlined letters represent the mutated residues of this study. *,
ligand to T3 copper; , ligand to T1 copper.
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 |
EXPERIMENTAL PROCEDURES |
Materials, Methods, and Enzyme Assays--
Chemicals used as
buffers and substrates were commercial products of at least reagent
grade. Britton and Robinson buffer (pH 2.7-11, made by mixing 0.1 M boric acid, 0.1 M acetic acid, and 0.1 M phosphoric acid with 0.5 M NaOH to desired
pH) was used for the pH profile. Recombinant TvL (isoform-1) was
purified as reported previously (16). The protocols for molecular
biology experiments (including restriction digests, DNA ligations, gel electrophoresis, and DNA preparations) were adapted from either the
instruction of the manufacturer or standard procedures.
Oligonucleotides were synthesized by an Applied Biosystems model 294 DNA/RNA synthesizer. Nucleotide sequences were determined by an Applied
Biosystems automatic DNA sequencer, model 373A-1.2.0. EPR spectra were
obtained using a Bruker ER 220-D-SRC spectrometer. All samples were run at 77 K in a liquid nitrogen finger Dewar flask. Spectrometer settings
were 10 milliwatts (or 13 decibels) power, 500 ms time constant, 20 Gauss (G) modulation, 1100 G sweep width, and 3050 G center field. The
ground state spin Hamiltonian parameters g
, g
, and A
were
obtained from simulations using the Quantum Chemistry Program Exchange
(QCPE) computer program "sim 15." Atomic absorption spectroscopy
was performed on a Perkin-Elmer 2380 instrument. The UV-visible
absorption spectra were recorded on a Shimadzu UV160U spectrophotometer
with 1-cm quartz cuvette. The extinction coefficient at 280 nm was
determined by amino acid analysis. The E° of T1 copper in
laccase was measured as reported previously (17) using the redox
titrant couple
Fe(2,2'-dipyridyl)2Cl3/Fe(2,2'-dipyridyl)2Cl2. Laccase-catalyzed oxidation of syringaldazine (SGZ) or
2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and
kinetic analysis were performed as reported previously (18). These two
substrates were chosen (to assay the activity of the mutants) because
of the two classes of the pH activity profiles that they possess
(19).
Preparation of TvL Mutants--
First, a 1.5-kilobase
BamHI-HindIII fragment encoding the T1
copper-ligating peptide segment of TvL was subcloned from pDSY10 (16)
into pUC118 (20). Second, a single-stranded DNA was prepared from this
intermediate and used as a template for site-directed mutagenesis (21)
with the primers 5'-dCTCGAGGCCGGCCTCGCCGTCGTG-3' (for F463L mutant),
5'-dGACTTCCACCTCTCGGCCGGCTTC-3' (for E460S mutant),
5'-dCACCTCGAGGAGGGCCTCGCCGTCGTG-3' (for A461E/F463L mutant), and
5'-dCTCGAGGCCGGCATGGCCGTCGTG-3' (for F463M mutant). The mutants were
identified by hybridization with the corresponding radiolabeled oligonucleotide primer and verified by DNA sequence analysis. The DNA
segments encoding TvL mutants were reinserted into pDSY10, then used to
transform an Aspergillus oryzae host. The transformation was
carried out by a procedure previously described (18). Cultures producing high levels of extracellular laccase activity were fermented in a laboratory-scale fermentor. The purification of the recombinant laccases was similar to that described previously (16). Recovery yield
of 15, 8, and 24%, and 300-, 540-, and 40-fold purification were
obtained for the F463L, A461E/F463L, and F463M mutants, respectively. Overall, the transformation and expression of the F463M, F463L, and
A461E/F463L mutants were comparable to the wt TvL, and the expressed
three mutants exhibited similar chromatographic elution patterns to the
wt TvL. Because of the lack of detectable laccase activity in shaker
flask, the E460S mutant was not purified and characterized.
 |
RESULTS AND DISCUSSION |
F463L Mutant and A461E/F463L Double Mutant--
The purified
mutants each had an UV-visible spectrum quite similar to the wt TvL
(Fig. 2, Table
II). The mutant enzymes had ~ 4 copper/protein and ~ 50% paramagnetism. The EPR spectrum of the
F463L mutant was obtained and showed a slight increase in g
and decrease in A
at the T1 site and a small change in g
of the
T2 copper site (Fig. 3, Table II).
However, these changes were minor, indicating that the F463L mutation
did not significantly alter the electronic structure of the T1 site. In 10 mM MES, pH 5.5, F463L exhibited an E°(T1
copper) that was only 0.05 V lower than that of the wt TvL (Table
III). This suggests that changing the
non-coordinating, bulky,
electron-rich Phe to Leu did not have a
dramatic impact on the T1 copper site. This result is consistent with a
previous study in which mutation of Leu to Phe in Rhizoctonia
solani and Myceliophthora thermophila laccases did not
result in alteration of the EPR parameters and E° for
their T1 copper (18). Table III and Fig.
4 summarize the SGZ and ABTS oxidase
activities of both the mutants. Km, kcat, and optimal pH (pHopt) were
not significantly altered in either the single or the double mutant.
The lack of a significant effect of these mutations on the electronic
properties of the T1 copper is consistent with the lack of an impact on
the kinetic parameters of the enzyme. Previous studies have shown that,
under steady state conditions, the rate-limiting step in the catalytic cycle is most likely the oxidation of the substrate by transfer of an
electron from the substrate to the T1 copper (22). If the electronic
structure of the T1 copper were significantly altered by a
mutation-induced perturbation of the site, it would seem likely that
the rate of substrate oxidation would also be affected.

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Fig. 2.
UV-visible spectra of TvL. A,
wt; B, F463L mutant; C, F463M mutant. The for
the 330-800 nm region has been magnified 16× to enable viewing.
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Table III
Redox potential and substrate specificity of the mutants
Redox potential and substrate specificity were measured in 10 mM MES, pH 5.5. ND, not determined.
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Fig. 4.
The pH activity profile for substrate SGZ
(A) and ABTS (B). Laccase used:
wt ( ); F463L (×); F463M ( ). Substrate concentration: SGZ, 19 µM; ABTS, 2 mM.
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F463M Mutant--
The transformation and expression of the F463M
mutant was comparable to that of the wt TvL. The expressed mutant
exhibited a chromatographic elution pattern similar to that of the wt
TvL. Copper content measurements revealed a stoichiometry number close to 4 copper/protein for the wt and mutant. The purified protein had an
UV-visible absorption spectrum characteristic of laccase (Fig. 2, Table
II). The absorption band at 600 nm had a maximal absorption wavelength
(
max) shifted 13 nm lower in energy, and the shoulder
around 750 nm is more pronounced, indicating that the mutation
perturbed the T1 copper. To further assess the extent of structural and
electronic changes of the T1 and T2 copper centers, we obtained the EPR
spectra for the mutant and wt enzymes (Fig. 3) and simulated the
spectra to extract the ground state spin Hamiltonian parameters (Table
II). The T1 copper site of the F463M mutant exhibited an increased
g
(2.214 as compared with 2.194 in the wt
enzyme) and a decreased A
(78 × 10
4 cm
1 as compared with 90 × 10
4 cm
1 for the wt enzyme). The T2 copper
remained unaltered. These changes are consistent with the T1 copper
site becoming more like the site in Pc and Rhus vernicifera
laccase (RvL). The EPR spectrum of Pc is characterized by a
g
of 2.226 and an A
of 63 × 10
4 cm
1, and RvL has a T1
copper with g
of 2.30 and
A
of 43 × 10
4
cm
1. While there is no sequence available for RvL, the
enzyme is believed to have a ligating Met in the axial position of the
T1 copper site, a structure analogous to that of Pc and zAO (Fig. 1).
Both the absorption and the EPR results indicate that the mutation led
to a significant change in the electronic structure of the T1 copper in
TvL.
In 10 mM MES, pH 5.5, the mutant exhibited an
E° 0.1 V lower than that of the wt TvL (Table III). It has
been proposed that the additional Met ligand would stabilize the
oxidized Cu(II) over the reduced Cu(I) site, thus leading to a lower
E° (3, 10, 11). Our EPR and absorption data strongly
suggested that the introduction of Met significantly perturbed the T1
site and that Met probably coordinated to the T1 copper based on the
spectral similarities to Pc. The decrease in the E° was
also consistent with this interpretation. The extent of the
E° change (0.1 V) agrees with previous studies on the T1
copper site in azurin in which the reverse mutation was carried out;
replacement of Met by a non-ligating Leu led to a 0.1-V increase in
E° (13, 14). These observations support the hypothesis
that ligation of the axial amino acid can tune the E° to
some extent. However it is important to note that the E°
of 0.68 V observed for the F463M mutant is still 0.1-0.4 V higher than
that of zAO, human serum ceruloplasmin, and Myrothecium
verrucaria bilirubin oxidase, whose T1 copper coordination also
includes the axial Met (3, 4, 17). Thus, other factors such as hydrogen
bonding, solvent accessibility, and orientation of local dipoles
clearly play an important role in tuning the E° (4, 23).
The pH profiles of the E° for the wt and F463M mutant are
depicted in Fig. 5. The profile of both
enzymes changed very little between pH 4 and 8 but showed an increase
at pH > 8. This increase was more pronounced in the F463M mutant.
It has been reported that the E° of Pc and azurin
increases as the pH decreases, due to the titration of a T1
copper-ligating H in the reduced site (24). The E° (T1) of
RvL also increases when pH decreases (25). Thus for TvL, the pH effect
on E° was in the opposite direction. Fig. 5 also shows the
pH profile of
max. No change was observed in either the
wt or F463M mutant over pH 2 through 8 but at pH > 8, a
significant decrease in
max was observed. This change in
max indicates a pH-dependent structural
change of the T1 copper site. While further spectroscopic studies are
needed to elucidate the origin of this change, it may be noteworthy
that the decrease in
max coincides with the increase in
E°.
The F463M mutation led to a 5-fold increase in Km
for the SGZ substrate and a 38-fold increase in Km
for the ABTS substrate (at pH 5.5). The mutation also resulted in a
2-fold increase in kcat (Table III). For an
electron-transfer reaction (such as the one between the substrate and
the T1 copper), its rate (kET) is determined by
thermodynamic driving force (E° difference between donor
and acceptor), electronic coupling (orbital overlap between donor and
acceptor), and Frank-Condon barrier (reorganization energy associated
with electron transfer) (26). The parameter kcat
is reflective of the rate-limiting step that was shown to be the
substrate oxidation under steady state conditions (22). While a number
of steps may contribute to kcat, the fact that
kcat was strongly correlated with the difference
in E° between the substrate and the T1 copper for a series
of substrates suggests that kET is a major
component of kcat. Our data illustrate an increase in kcat of substrate oxidation that can
be explained by an increase in kET. The
decreased E° of the T1 copper in the F463M mutant would
lead to a decreased thermodynamic driving force and thus a lower
kET. Thus, in order to account for the increase in kcat the mutation must have affected the
electronic coupling and/or the reorganization energy. The EPR spectrum
of F463M provides initial evidence that the mutation caused a change in
the ground state of the T1 copper. This could result in a change in the
orbital overlap between the donor (substrate) and acceptor (the half
occupied highest molecular orbital of the T1 copper). There is some
evidence from ENDOR that the spin density on the imidazole nitrogen
ligands of different T1 copper sites can vary, depending on the nature of the ground state (27). This difference could influence the orbital
overlap between the donor and acceptor. It is also possible that the
mutation altered the Frank-Condon barrier to electron transfer.
Evaluation of electronic structure differences between wt and F463M,
which would relate to this difference in reactivity, is presently under way.
The F463M mutation led to a shift in pHopt for the
oxidation of SGZ but not ABTS (Fig. 4). It has been shown that
oxidation of a phenolic substrate depends on whether it is protonated;
the deprotonated phenol has a lower redox potential and therefore is
more easily oxidized (19). The fact that the pH dependence of activity
was only altered when SGZ, a phenolic substrate, was used but not when
ABTS, a non-phenolic substrate, was used indicates that the mutation
must have caused a slight change in the substrate-binding pocket. This
is also consistent with the change in Km and could
perturb the protonation equilibrium of the phenolic substrate.
Concluding Remarks--
Understanding how the E° of
copper sites in proteins is regulated and how E° and
geometric and electronic structure perturbations influence the electron
transfer function of a protein is one of the major challenges in the
field of metallo-biochemistry. Various theories have been proposed to
explain how two seemingly similar T1 copper sites could have different
E° and different reactivity (1-4, 17). For these
comparisons, it is important to obtain a detailed description of the
electronic structure of the copper in a similar environment. Our
results provide initial evidence that a more "classic" blue copper
site can be created in fungal laccase through site-directed mutagenesis
and that the resulting electronic structure appears to be intermediate
between the classic Pc T1 copper site and that of the wt fungal
laccase, which has no axial Met. This leads to a lowering of
E° in the same protein environment consistent with the
axial Met stabilizing the oxidized, Cu(II) state more than the reduced,
Cu(I) state. Detailed spectral studies are currently being pursued to
understand the effects of the associated electronic structure changes
on the reactivity of these enzymes.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Alan V. Klotz, Glenn E. Nedwin,
Anders H. Pedersen, and Ejner B. Jensen of Novo Nordisk for critical
reading and helpful suggestions, as well as Kimberly M. Brown,
Michael W. Rey, Elizabeth J. Golightly, and Sheryl Bernauer of
Novo Nordisk Biotech for carrying out protein/DNA sequencing.
 |
FOOTNOTES |
*
This work was partially supported by National Institutes of
Health Grant DK 31450 (to E. I. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence may be addressed: Novo Nordisk Biotech, 1445 Drew Ave., Davis, CA 95616. Tel.: 530-757-8100; Fax: 530-758-0317; E-mail: fengxu{at}nnbt.com.
To whom correspondence may be addressed: Dept. of Chemistry,
Stanford University, Stanford, CA 94305. Tel.: 650-723-9104; Fax:
650-725-0259; E-mail:
fb.eis{at}forsythe.stanford.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
T1, type 1 copper;
T2, type 2 copper;
T3, type 3 copper;
TvL, recombinant T. villosa
(Polyporus pinsitus) laccase (isozyme 1);
RsL, recombinant
R. solani laccase;
MtL, recombinant M. thermophila laccase;
CcL, recombinant C. cinereus
laccase;
PvL, T. (Polyporus) versicolor laccase;
zAO, Zucchini ascorbate oxidase;
RvL, R. vernicifera
laccase;
Pc, poplar plastocyanin;
wt, wild type;
ABTS, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid);
SGZ, syringaldazine;
E°, single-electron redox potential
(referenced to the normal hydrogen electrode) at T1 site;
pHopt, optimal pH;
G, gauss;
MES, 4-morpholineethanesulfonic acid..
 |
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