Báo cáo khoa học: Role of the C-terminal extension in a bacterial tyrosinase Michael Fairhead and Linda Thony-Meyer doc

Role of the C-terminal extension in a bacterial tyrosinase
Michael Fairhead and Linda Tho
¨
ny-Meyer
EMPA, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for Biomaterials, St Gallen, Switzerland
Introduction
Tyrosinases and the related catechol oxidases (collec-
tively termed polyphenol oxidases) comprise a family
of binuclear copper enzymes found in many species
of animals, plants, fungi and bacteria that use phe-
nol-like starting materials to produce a variety of
biologically important compounds, such as melanin
and other polyphenolic compounds [1–3]. These
type III copper proteins are capable of two activities:
monophenolase or cresolase activity (EC 1.14.18.1)
and diphenolase or catecholase activity (EC 1.10.3.1).
Both activities result in the formation of reactive
quinones, and these species are important intermedi-
ates in the biosynthesis of compounds such as
melanin.
Given the ability of tyrosinases to react with phenols
and its di-copper redox centres, they have been
proposed for use in a variety of biotechnological,
biosensor and biocatalysis applications [2]. One exam-
ple includes tyrosinase immobilization as an electro-
chemical biosensor for a range of phenolic compounds
[4]. The enzyme can also react with tyrosine found on
polypeptides, and the reactive quinones formed allow
for protein cross-linking to chitosan films as well as
protein-protein cross-linking [5,6].
The only available crystal structure of the tyrosin-

ases comes from the secreted enzyme of Streptomyces
castaneoglobisporus [7] tyrosinase. The structure shows
the enzyme in complex with its accessory caddie
protein (see below). The tyrosinase is predominately
a-helical in structure and contains six histidine residues
co-ordinating the two copper atoms that form the
active site of the enzyme. With respect to its overall
fold and active site architecture, the bacterial enzyme
is strongly similar to the related enzyme catechol
Keywords
C-terminal domain; melanin; tyrosinase;
Verrucomicrobium spinosum; zymogen
Correspondence
L. Tho
¨
ny-Meyer, EMPA, Swiss Federal
Laboratories for Materials Testing and
Research, Laboratory for Biomaterials,
Lerchenfeldstrasse 5, St Gallen, CH-9014,
Switzerland
Fax: +41 44 071 274 7788
Tel: +41 44 071 274 7792
E-mail:
(Received 22 October 2009, revised
13 January 2010, accepted 22 February
2010)
doi:10.1111/j.1742-4658.2010.07621.x
The well studied bacterial tyrosinases from the Streptomyces sp. bacteria
are distinguishable from their eukaryotic counterparts by the absence of a
C-terminal extension. In the present study, we report that the tyrosinase

from the bacterium Verrucomicrobium spinosum also has such a C-terminal
extension, thus making it distinct from the Streptomyces enzymes. The
entire tyrosinase gene from V. spinosum codes for a 57 kDa protein (full-
length unprocessed form), which has a twin arginine translocase type signal
peptide, the two copper-binding motifs typical of the tyrosinase protein
family and the aforementioned C-terminal extension. We expressed various
mutants of the recombinant enzyme in Escherichia coli and found that
removal of the C-terminal extension by genetic engineering or limited tryp-
sin digest of the pro-form results in a more active enzyme (i.e. 30–100-fold
increase in monophenolase and diphenolase activities). Further studies also
revealed the importance of a phenylalanine residue in this C-terminal
domain. These results demonstrate that the V. spinosum tyrosinase is a new
example of this interesting family of enzymes. In addition, we show that
this enzyme can be readily overproduced and purified and that it will prove
useful in furthering the understanding of these enzymes, as well as their
biotechnological application.
Abbreviations
L-DOPA, L-3,4-dihydroxyphenylalanine; TAT, twin arginine translocase.
FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2083
oxidase from sweet potato [8]; however, the plant
enzyme is only capable of the diphenolase reaction
(EC 1.10.3.1).
The major distinguishing feature of the Strepto-
myces sp. enzyme is the requirement for an accessory
protein that is necessary for copper incorporation [1].
Several mutagenesis studies, as well as the crystal
structure, have demonstrated the importance of this
accessory ‘caddie protein’ for copper incorporation
into the Streptomyces tyrosinase [7,9] and the expres-
sion of active Streptomyces tyrosinase in either

Escherichia coli or its native host requires the
co-expression of the gene encoding this caddie pro-
tein. This arrangement is entirely different from that
of the eukaryotic enzymes, which are not known to
require such a caddie protein and also have a C-ter-
minal extension, the removal of which usually leads
to a marked increase in activity [10]. Indeed, it is esti-
mated that approximately 98% of the the tyrosinase
present in mushrooms occurs in such a latent form
[11]. However, the Streptomyces sp. tyrosinases may
not be wholly representative of the bacterial form of
these enzymes because the Rhizobium etli tyrosinase
has been reported not to require a copper chaperone
for activity [12].
Given their interesting properties and the wide poten-
tial of these enzymes, there are few successful examples
of recombinant production systems that provide high
yields of pure enzyme, with most studies using the native
Streptomyces sp. [13,14], Neurospora crassa [15] and
Agaricus bisporus [2] enzymes. To cover this shortfall,
we have cloned several uncharacterized tyrosinase
genes from different bacterial species with the aim of
identifying enzymes that have suitable characteristics
for structure ⁄ function studies, as well as biotechnologi-
cal applications. In the present study, we report
the results obtained with the tyrosinase gene from
Verrucomicrobium spinosum.
Verrucomicrobium spinosum is part of the ubiquitous
Verrucomicrobia phylum. These bacteria are found in
a wide range of aquatic and terrestrial habitats

[16,17]. Verrucomicrobium spinosum in particular is
found in fresh water eutrophic (nutrient rich, oxygen
poor) habitats and is capable of both aerobic and fer-
mentative metabolism. This Gram-negative, yellow-
pigmented bacterium is somewhat unusual as a result
of the presence of numerous wart-like prosthecae
appendages on its surface [17,18] and its compartmen-
talized cytoplasm [19]. This bacterium is not known to
normally produce melanin, and thus the presence of a
tyrosinase gene in its genome was somewhat surprising
because such genes are usually associated with
black pigment formation in various bacterial and
fungal species [20].
Results and Discussion
Analysis of the V. spinosum tyrosinase gene
region
The V. spinosum tyrosinase gene is preceded upstream
by a gene encoding a predicted laccase and followed
downstream by a gene encoding a predicted b-sheet-
rich protein for which we could find no obvious func-
tion or homologue (Fig. 1A). This differs from the
Streptomyces tyrosinase gene arrangement, where the
tyrosinase is typically preceded by a gene encoding an
accessory protein required for copper incorporation
[1]. Given the absence of such a caddie protein gene
upstream or downstream of the V. spinosum tyrosinase
gene, we drew the conclusion that the V. spinosum
tyrosinase does not require such a protein for copper
insertion. The V. spinosum tyrosinase may therefore be
similar to the aforementioned R. etli tyrosinase, which

also has been reported not to require a copper chaper-
one [12]. The presence of another multicopper oxidase-
like laccase gene upstream of the tyrosinase gene
is also interesting because laccases are known to be
capable of synthesizing melanin, albeit usually from
diphenols such as epinephrine and l-3,4-dihydroxy-
phenylalanine (l-DOPA) [21].
Also present in the surrounding DNA sequence are
several regions with homologies to the binding sites of
E. coli RpoS and RpoD sigma factors, which are
known to be involved in transcriptional regulation
[22]. The predicted b-sheet-rich protein gene is fol-
lowed by a region with a high probability of leading to
an RNA secondary structure in the transcript, indica-
tive of a site of transcription termination. The presence
of these features may indicate that the tyrosinase gene
is part of an operon.
As stated in the Introduction, V. spinosum is not
known to produce melanin under normal growth
conditions. The laccase and ⁄ or tyrosinase are there-
fore probably only synthesized under a specific set of
circumstances or serve some alternative function to
melanin production. We attempted to induce melanin
synthesis by cultivating the V. spinosum bacterium on
solid or in liquid media supplemented with excess
copper or amino acids in an attempt to mimic con-
ditions known to induce Streptomyces species tyro-
sinases [23]. However, these experiments did not
yield any detectable tyrosinase activities, as indicated
by the lack of formation of any black pigments or

Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho
¨
ny-Meyer
2084 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS
monophenolase ⁄ diphenolase activities in bacterial
extracts (data not shown).
Features of the amino acid sequence of the
V. spinosum tyrosinase
The amino acid sequence of the full-length V. spino-
sum pre-pro-tyrosinase (Fig. S1) can be divided
approximately into three domains: a twin arginine
translocase (TAT) signal peptide, a core domain con-
taining the two copper-binding motifs and a C-termi-
nal extension (Fig. 1B). The presence of a predicted
TAT signal peptide at the N-terminus (amino acids
1–36) would suggest that the protein is exported to the
periplasmic space of V. spinosum in an already folded
form, as often found for metal-containing periplasmic
proteins [24]. The presence of this signal peptide is in
agreement with the fact that the Streptomyces tyrosin-
ases are also secreted via the TAT secretion pathway
[25].
Also present in the sequence are the two copper
A (amino acids 86–96) and copper B (amino acids
258–294) binding motifs common to most tyrosinase
sequences [3] that contain five of the six copper-bind-
ing histidine ligands. The sixth histidine ligand found
in tyrosinases typically occurs before the copper A
motif. From sequence alignments, we suggest that this
ligand is most likely histidine 80 in the V. spinosum

tyrosinase. Another motif, which is present not only in
tyrosinases, but also in the oxygen transporting
haemocyanin proteins, is the PYWDW (amino acids
118–122) and has been hypothesized to be involved in
oxygen binding [26].
Previous sequence analysis in other studies has dem-
onstrated the presence of a conserved Yx(Y ⁄ F) motif
in the C-terminal domains of both the Streptomyces
type tyrosinases and processed eukaryotic tyrosinases
and haemocyanins [10]. This motif can also be seen to
be present in the V. spinosum tyrosinase (Figs 1B and
S1). It has been hypothesized, with support from the
crystal structure of catechol oxidase, that the tyrosine
residue(s) in this motif form a hydrogen-bonding
network to a conserved arginine residue close to the
N-terminus that stabilizes the mature, processed form
of polyphenol oxidases [8,10]. A homologue of
this arginine residue is also present in V. spinosum
tyrosinase (Arg40) (Figs 1 and S1).
Another notable feature of the V. spinosum tyrosi-
nase sequence is the presence of the proteins only cys-
teine residue at position 84. A cysteine at this position
is also found in some other eukaryotic tyrosinases and
plant catechol oxidases. This cysteine may be of
functional importance because it has been shown to
form a novel alkane-thiol bond to one of the copper
ligand histidine residues in the structure of the related
sweet potato catechol oxidase [8]. The equivalent
cysteine and bond are absent in the structure of S. cas-
taneoglobisporus tyrosinase [7]. Indeed, Streptomyces

A
Copper
binding
motif
TAT signal peptide
1–36
Pre-pro-tyrosinase 518 amino acids
Core domain
37–357
C-terminal extension
amino acids 358–518
Copper
binding
motif
Arg40
Phe453
Cys84
Tyr349
Tyr347
Copper
binding
motif
Pro-tyrosinase 481 amino acids
Core domain
36–357
C-terminal extension
amino acids 358–518
Copper
binding
motif

Arg40
Phe453
Cys84
Tyr349
Tyr347
Copper
binding
motif
Core tyrosinase 320 amino acids
Core domain
36–357
Copper
binding
motif
Arg40
Cys84
Tyr349
Tyr347
Copper
binding
motif
Trypsinisedpro-tyrosinase 332 amino acids
Core domain
36–370
Copper
binding
motif
Arg40
Cys84
Tyr349

Tyr347
Lys370
Ala36
Ala36
Ala36
Val357
Phe518
B
Laccase
Tyrosinase
β
-sheet protein
Fig. 1. Overview of the tyrosinase gene
and surrounding genes in the genome of
V. spinosum. (A) Showing the tyrosinase
gene and those in its immediate vicinity in
the V. spinosum genome. Triangles indicate
regions with homology to the binding sites
of the E. coli RpoS and RpoD regulatory
proteins; the octagon shows the position of
a region predicted to have a high probability
of RNA secondary structure, which is
indicative of a termination transcript.
(B) An overview of the pre-pro-tyrosinase,
pro-tyrosinase and core-tyrosinase
constructs and their notable features.
M. Fairhead and L. Tho
¨
ny-Meyer Recombinant V. spinosum tyrosinase
FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2085

sp. tyrosinases contain no cysteine residues at all [27].
However, experimental evidence does demonstrate the
presence of such a bond in N. crassa tyrosinase [15]
and in molluscan haemocyanins [28].
The arrangement of a core tyrosinase domain
followed by a C-terminal extension (Fig. 1B) is similar
in design to mushroom tyrosinase and plant polyphenol
oxidases [10]. The mushroom C-terminal domain can be
removed by proteolysis or denatured by SDS, leading to
an activation of the enzyme [11,29]. By contrast, the
Streptomyces type tyrosinases have no such C-terminal
extension after the core tyrosinase domain [1].
One proposed function of the C-terminal extension
in plant and fungal polyphenol oxidases is a role in
membrane binding, making them similar to the mam-
malian tyrosinases, which have a single transmembrane
domain [27]. However, it is considered that the plant
forms are not integral membrane proteins because they
can be released in an active form from the membrane
by sonication, proteolysis or treatment with mild deter-
gents [30,31]. Thus, whether the C-terminal domain in
the plant and fungal enzymes has a purely inhibitory
function and ⁄ or a role in membrane binding is unclear
at present. With regard to V. spinosum pro-tyrosinase,
sequence analysis of the C-terminal domain, and
indeed of the entire sequence, suggested that no trans-
membrane helices were present, as also demonstrated
by the fact the enzyme is produced in a soluble form
in E. coli.
Recombinant expression of V. spinosum

tyrosinase in E. coli
To study the properties of the V. spinosum tyrosinase,
we created a range of constructs (Table 1) for recombi-
nant expression of the pre-pro-tyrosinase, the pro-
tyrosinase and the core tyrosinase (Fig. 1B). It can be
seen from Fig. 2 that E. coli cells transformed with
plasmids containing either the pre-pro-tyrosinase or
the pro-tyrosinase tyrosinase constructs (Fig. 2B, C)
produced a black pigment when streaked onto M9
agar plates containing tyrosine and copper, whereas a
strain lacking a tyrosinase construct remained white
(Fig. 2A).
The activity observed on the M9 agar plates was
found to correlate with over-expression of the various
proteins in liquid media. It can be seen from the gel
presented in Fig. 3A that bands are present in samples
of lysate of E. coli cells transformed with plasmids
encoding the different tyrosinase variants. These bands
correspond to the calculated molecular masses of the
respective polypeptides (Table 1), namely 57 kDa for
pre-pro-tyrosinase (lane 4) and 53.4 kDa for pro-tyros-
inase (lane 3). The different constructs were expressed
at different levels, with an increase in expression occur-
ring when the putative N-terminal TAT signal peptide
was removed (Fig. 3A, lanes 3 and 4).
We found it necessary to express all the tyrosinase
constructs in an apo-form, by growing and inducing
Table 1. List of active constructs produced in the work and their features. ND, not determined; NA, not applicable.
Name (plasmid)
Mutations or

modifications
Calculated
molecular
mass
(kDa)
a
Determined
molecular
mass
b
pI
a
Extinction
coefficient
280 nm
(m
M
)1
Æcm
)1
)
a
Purpose
Pre-pro-tyrosinase
(pMFvppt)
Amino acids 1–518 57.005 ND 7.2 91.9 Full-length tyrosinase
gene from
V. spinosum
Pro-tyrosinase
(pMFvpt)

Amino acids 36–518 with
non-original methionone
start codon
53.500 53.501 6.9 86.4 Removal of TAT signal
pepetide from
pro-tyrosinase gene
for cytosolic expression
Trypsinized
pro-tyrosinase (NA)
Amino acids 36–370 37.873 37.874 8.1 80.9 Removal of c-terminal
extension via trypsin
for improved activtiy
Core tyrosinase
(pMFvct)
Amino acids 36–357 with
non-original methionone
start codon
36.507 36.506 7.1 80.9 Removal of c-terminal
extension for improved
activity
Pro-tyrosinase
F453A (pMFvptf2a)
Pro-tyrosinase with
phenylalanine 453
mutated to alanine
53.4 ND 6.9 86.4 To check whether this
residue performs a
‘gatekeeper’ function at
the tyrosinase active site
a

Values calculated using PROTPARAM (24).
b
Molecular mass determined by MS.
Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho
¨
ny-Meyer
2086 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS
the transformed cells in media prepared using Milli-Q
water (Millipore, Billerica, MA, USA) and lacking
added copper. This was necessary because, otherwise,
a black pigment was produced during incubation. This
pigment was found to inhibit the growth of E. coli and
to foul protein purification columns, both of which
resulted in a low protein yield. This problem was par-
ticularly acute with the highly active core tyrosinase.
The formation of a black pigment (presumably mela-
nin) was most likely a result of the action of the
expressed tyrosinase on the tyrosine present in the pep-
tone or N-Z-amine
Ò
(Sigma-Aldrich, Buchs, Switzer-
land) that was added to the expression medium as an
external source of amino acids to aid recombinant
protein production. Provided the precaution of not
supplying copper to the medium was taken, we found
that soluble protein could be obtained for all the
described constructs.
In experiments with the pre-pro-tyrosinase construct,
we did not obtain sufficient amounts of protein for
purification. We also attempted to isolate the protein

from the E. coli periplasm but could not find any evi-
dence of activity, indicating a lack of export of the
protein. It could be that the E. coli TAT system is
unable to recognize the V. spinosum export signal
peptide.
When designing tyrosinase constructs without the
predicted N-terminal signal peptide (amino acids
1–36), we retained amino acid 36, an alanine, rather
than using amino acid 37, a lysine, because it is
known that, after a post-translational processing of the
N-terminal methionine, which often occurs for proteins
expressed in E. coli, according to the N-end rule, a
newly-created N-terminal lysine would result in a very
short protein half-life, whereas an N-terminal alanine
would be fine [32].
The recombinant pro-tyrosinase was expressed and
purified with final yields of approximately 20 mgÆL
)1
of pure protein. Subsequent analytical gel filtration of
the purified pro-tyrosinase showed a single peak corre-
sponding to a monomer (Fig. S2). The mass of the
purified protein determined via MS (53 501 kDa)
corresponded closely to the expected full-length pro-
tyrosinase (53 500 kDa) assuming the removal of the
N-terminal methionine.
Reconstitution of recombinat V. spinosum
tyrosinase with copper
The holo-forms of tyrosinase were obtained after puri-
fication by adding copper to a three-fold molar excess,
and samples were subsequently exhaustively dialysed in

an attempt to remove any nonspecifically bound cop-
per. The final copper content of the dialysed samples
was then determined (Table 2). Although pro-tyrosi-
nase was found to be nearly fully loaded with copper
using this method (1.8 molar equivalents), the core
tyrosinase and pro-tyrosinase F453A mutant were
found to be significantly under-loaded (1.4 and
1.2 molar equivalents respectively). It is possible
that the protocol used was not optimal for copper
incorporation into these variants (see Experimental
A
CD E
B
Fig. 2. Melanin formation on tyrosine con-
taining solid media by E. coli cells express-
ing V. spinosum tyrosinase constructs. (A)
Escherichia coli transformed with vector
containing no insert (pQE-60); (B) E. coli
transformed with pMFvppt (pre-pro-tyrosi-
nase); (C) E. coli transformed with pMFvpt
(pro-tyrosinase); (D) E. coli transformed with
pMFvct (core tyrosinase); (E) E. coli trans-
formed with pMFvptf2a (pro-tyrosinase
F453A).
M. Fairhead and L. Tho
¨
ny-Meyer Recombinant V. spinosum tyrosinase
FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2087
procedures) and, indeed, it has been reported that
incubation at pH 6 may result in higher levels of cop-

per reconstitution than at pH 8 [33,34]. We are cur-
rently investigating this possibility.
In addition, despite extensive dialysis of reconsti-
tuted samples, it cannot be excluded that some of the
copper is nonspecifically bound to the protein. We
have found, however, that attempts to remove any
such copper ions with low concentrations of the chelat-
ing agent EDTA (100 lm) resulted in a complete loss
of activity and detectable copper. As an alternative to
copper reconstitution of the purified proteins, we also
attempted to grow bacteria in minimal media contain-
ing copper as a means of producing holo protein
directly. However, we found that the omission of an
external amino acid source such as N-Z-amine led to
very low levels of tyrosinase expression, as well as low
cell densities, meaning that the purification of holo
protein in this way was impracticable.
C-terminal processing by trypsin
As noted above, the C-terminal extension found in the
latent form of mushroom tyrosinase has been shown
to be inhibitory to activity, and its removal by serine
proteases such as subtisilin results in an activation of
the enzyme, similar to the protease zymogen system
found for many digestive enzymes, such as trypsin [11].
The related plant catechol oxidase enzymes also have
similar C-terminal extensions [10]. Sequence analysis
suggested that this may also be the case for the
V. spinosum enzyme (see above). We therefore used
trypsin digestion to determine whether a smaller, more
active fragment could be produced from purified pro-

tyrosinase. The gel in Fig. 3C shows that trypsin diges-
tion indeed yielded a smaller stable fragment, which
was subsequently found to be far more catalytically
active than the original pro-tyrosinase (Table 3). The
stability of the smaller trypsinized fragment, even after
24 h of incubation with trypsin, suggests that this is a
highly ordered domain with no accessible cleavage sites
for trypsin. This interpretation corresponds to the pro-
posal that the C-terminal extension of eukaryotic poly-
phenol oxidases (i.e. tyrosinase and plant catechol
oxidases) is highly disordered [10] compared to the
corresponding core oxidase domains containing the
two copper-binding motifs. These disordered domains
would thus be more susceptible to proteolysis than the
more ordered stable core domains of the enzymes.
High levels of disorder in the pro-domain are also
present in zymogens such as in procathepsin K [35]
and probably represent an important feature in the
activation mechanism of these enzymes. The fact that
A
B
C
Fig. 3. (A) SDS-PAGE of cells expressing the tyrosinase constructs.
Lane 1, lysate from cells transformed with pMFvptf2a (pro-tyrosi-
nase F453A); lane 2, lysate from cells transformed with pMFvct
(core tyrosinase); lane 3, lysate from cells transformed with pMFvpt
(pro-tyrosinase); lane 4, lysate from cells transformed with pMFvppt
(pre-pro-tyrosinase); lane 5, lysate from control cells transformed
with pQE-60 containing no insert. (B) SDS-PAGE of purified and
trypsinized tyrosinases. Lane 1, purified pro-tyrosinase; lane 2, puri-

fied core tyrosinase; lane 3, purifed pro-tyrosinase F453A mutant;
lane 4, trypsinized pro-tyrosinase; lane 5, trypsinized core tyrosi-
nase. (C) SDS-PAGE showing time course of proteolysis of
pro-tyrosinase by trypsin. Lane 1, pro-tyrosinase after 24 h of incu-
bation at room temperature; lane 2, trypsin after 24 h of incubation
at room temperature; lane 3, pro-tyrosinase plus trypsin after 0 h
at room temperature; lane 4, pro-tyrosinase plus trypsin after 1 h at
room temperature; lane 5, pro-tyrosinase plus trypsin after 4 h
at room temperature; lane 6, pro-tyrosinase plus trypsin after
24 h at room temperature. M, Molecular mass markers.
Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho
¨
ny-Meyer
2088 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS
the pro-tyrosinase exhibits some low levels of catalytic
activity also suggests some mobility between the core
tyrosinase domain and the C-terminal extension
(Table 3).
Recombinant core tyrosinase
To further asses the functional importance of the
C-terminal extension, we created a shortened form of
the V. spinosum tyrosinase, using the presence of the
conserved YX(Y ⁄ F) motif as a guide. The resulting
construct was readily overexpressed in the E. coli
cytoplasm (Fig. 3A, lane 4) and found to be highly
active after loading with copper compared to the
pro-tyrosinase form (Table 3).
We also treated the mature (i.e. copper-containing)
tyrosinase with trypsin and found that the trypsinized
recombinant core tyrosinase (Fig. 3B, lane 5) exhibited

no apparent size difference compared to the un-trypsi-
nized preparation (Fig. 3B, lane 2) but appeared to be
smaller than the trypsinized pro-tyrosinase (Fig. 3B,
lane 4). Determination of the mass of the proteins by
MS revealed masses of 36 506 kDa (recombinant core
tyrosinase) and 37 874 kDa (trypsinized pro-tyrosi-
nase) corresponding to a C-terminal amino acid of
Val357 and Lys370, respectively. Gel filtration revealed
that both proteins also exist in solution, similar to pro-
tyrosinase, as monomers (Fig. S2).
The results obtained in the present study suggest
that the C-terminal extension has no role in copper
insertion like the Streptomyces sp. ‘caddie’ protein
because the recombinant core tyrosinase enzyme was
found to be readily reconstituted with copper, as indi-
cated by its high activity and subsequent analysis of its
copper content (Table 2). This correlates with the
results obtained using apo-forms of mature tyrosinase
from both N. crassa [36] and A. bisporus [37], which
could also be readily reconstituted with copper. This is
in contrast to the results obtained with the Streptomy-
ces sp. enzyme [38,39], which has an absolute require-
ment for the accessory caddie protein for copper
incorporation. Furthermore, the results obtained in the
present study are in agreement with the previously
noted finding that, in the gene region around the
V. spinosum tyrosinase, no gene encoding a caddie-like
protein is present (Fig. 1A).
Because the pro-tyrosinase form contains no
predicted transmembrane helices and is indeed fully

soluble in E. coli (see above), we suggest that the
C-terminal extension in this case has a purely inhibitory
function and neither a significant role in stabilizing the
enzyme, nor a chaperone-like function during folding,
as has been proposed for other N-terminal ⁄ C-terminal
zymogen-like systems [40]. It remains to be determined
whether this is also the case for other pro-tyrosinase
forms.
Stability of the tyrosinase forms to chemical
denaturation
To characterize the domain structure of the V. spino-
sum tyrosinase in more detail, we determined protein
stability by recording protein unfolding via fluores-
cence spectroscopy when increasing amounts of guani-
dine hydrochloride (GdnCl) were present. The
determined unfolding curves (Fig. S3) appeared to
show two apparent transitions for holo pro-tyrosinase
and one for either holo trypsinized pro-tyrosinase or
the holo recombinant core tyrosinase. However, the
unfolded proteins were not found to refold once
Table 2. Stability and determined copper content of the tyrosinase
enzymes. ND, not determined.
Enzyme
GdnCl
concentration (
M)
at 50% unfolded
a
Molar
equivalents

of copper
Holo pro-tyrosinase 2.2 1.8
Apo pro-tyrosinase 1.3 0.01
Holo trypsinized pro-tyrosinase 3.3
b
1.8
b
⁄ 1.5
c
Apo tyrpsinized pro-tyrosinase 2.0 0.4
Holo core tyrosinase 2.9 1.4
Apo core tyrosinase 1.8 0.02
Holo pro-tyrosinase F453A ND 1.2
Apo pro-tyrosinase F453A ND 0.1
a
Protein solutions (0.1 mgÆmL
)1
) were incubated for 24 h at room
temperature in 10 m
M Tris-HCl (pH 8) containing 0–6 M GdnCl
before measurements were made (for details, see Experimental
procedures).
b
Copper content and stability determined with trypsi-
nized holo pro-tyrosinase.
c
Copper content determined by reconsti-
tuting trypsinized apo pro-tyrosinase.
Table 3. Monophenolase and diphenolase activities of the tyrosi-
nase enzymes. Activity of the various constructs ⁄ mutants towards

the model substrates
L-tyrosine and L-DOPA (n = 3 for all determi-
nations).
Enzyme
L-tyrosine L-DOPA
V
max
a
K
m
(lM) V
max
a
K
m
(mM)
Pro-tyrosinase 5.8 ± 0.6 421 ± 43 4.7 ± 0.3 7.0 ± 0.7
Trypsinized
pro-tyrosinase
b
325 ± 8 258 ± 6 565 ± 20 7.9 ± 0.5
Core tyrosinase 148 ± 4 280 ± 15 230 ± 7 7.6 ± 0.3
Pro-tyrosinase
F453A
16 ± 0.9 808 ± 66 14 ± 0.2 6.4 ± 0.4
a
Units = lmol dopachromeÆmin
)1
ÆmgÆprotein
)1

.
b
Values deter-
mined for trypsinized holo pro-tyrosinase.
M. Fairhead and L. Tho
¨
ny-Meyer Recombinant V. spinosum tyrosinase
FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2089
denatured and, thus, the apparent shapes of the
unfolding curves should not be over interpreted. The
use of the concentration of GdnCl at 50% unfolded as
a simple measure of the change in stability between the
various tyrosinase forms allows some conclusions to be
drawn (Table 2). The values show that the incorpora-
tion of copper into either pro-tyrosinase, trypsinized
pro-tyrosinase or recombinant core tyrosinase signifi-
cantly increases the overall stability of the protein. It
was also apparent that the C-terminal extension of
pro-tyrosinase reduces its overall stability in either the
holo- or apo-forms of the enzyme. The negative effect
on stability as a result of C-terminal extension would
suggest this domain is less stable than the core domain
of the enzyme, which correlates with the results
obtained with trypsin digestion. It can also be seen
from Table 2 that the recombinant core domain tyrosi-
nase appears to be less stable than the trypsinized pro-
tyrosinase; this could be a result of its reduced copper
content. Alternatively, it may be that the recombinant
core tyrosinase C-terminal extension is slightly too
short for optimal stability and that residues after the

YX(Y ⁄ F) motif also play a role in protein stability.
Mono- and diphenolase activities of the
recombinant tyrosinases
When we measured activities towards either l-tyrosine
or l-DOPA of pro-tyrosinase, a major increase in
activity upon removal of the C-terminal extension by
trypsin was found, namely an approximately 50-fold
increase in mono- and a 100-fold increase in dipheno-
lase activitiy (Table 3). There was also a less significant
lowering in the K
m
value for l-tyrosine upon removal
of the C-terminal extension (i.e. from 421 to 258 lm).
The activities of the trypsinized pro-tyrosinase
towards l-tyrosine or l-DOPA was found to be
approximately twice that of the recombinant core
tyrosinase, although the K
m
for both substrates is
almost identical. The increased level of activity is prob-
ably a result of the higher copper content of the trypsi-
nized pro-tyrosinase (Table 2). The actual activities of
the trypsinized pro-tyrosinase and recombinant core
tyrosinase towards l-DOPA (i.e. 565 and 230 lmol
dopachromeÆmin
)1
Æmg protein
)1
, respectively) compare
favourably with the activities reported for Strepto-

myces antibioticus tyrosinase, which are 1000 dopa-
chromeÆmin
)1
Æmg protein
)1
[41]. The K
m
values for
these two preparations towards l-DOPA (7.9 and
7.6 mm, respectively) are also similar to those report-
ed for the S. castaneoglobisporus enzyme (8.1 mm)
but substantially higher than that reported for the
A. bisporus enzyme (0.8 mm) [42]. However, the K
m
values for l-tyrosine (258 and 280 lm, respectively)
were similar to that of the A. bisporus enzyme
(270 lm) [42].
Role of Phe453 in the pro-tyrosinase C-terminal
The inhibitory effect of the C-terminal extension found
in some plant polyphenol oxidases has been hypothe-
sized to be a result of the presence of an amino acid
that occludes the active site. This idea has been
proposed because of similarities in the structures of the
C-terminals of the related family of haemocyanins to
plant polyphenol oxidases [3]. The crystal structure of
octopus haemocyanin shows that a leucine (Leu2830)
residue is present near the active site and acts as a
‘blocking residue’ [43]. This ‘blocking residue’ prevents
substrate molecules from entering the active site,
although oxygen can freely diffuse in and out, allowing

oxygen transport to be the primary function of this
protein. However, upon denaturation with SDS or
proteolysis, it has been observed that tyrosinase-like
activities can be introduced into haemocyanins and
this has been proposed to occur via movement of the
‘blocking residue’ [44]. A leucine or similar hydropho-
bic residue in an equivalent position has also been
demonstrated to be present by sequence alignments
of plant polyphenol oxidases [3]. In the case of the
catechol oxidase from Ipomea, molecular modelling of
the C-terminal domains was used to propose Leu439
as the ‘blocking residue’ [45].
Using a similar process of sequence alignment, we
hypothesized that the functional equivalent of this
blocking residue in V. spinosum pro-tyrosinase is
Phe453. Thus, we constructed a pro-tyrosinase mutant
carrying an alanine at this position, F453A. Curi-
ously, an increase in protein expression was obtained
for this mutant tyrosinase similar to that obtained
when the entire C-terminal extension was removed
(i.e. that of the core tyrosinase; Fig. 3B, lanes 1–3). It
can be seen from the results shown in Table 3 that
this variant had a higher activity than wild-type pro-
tyrosinase, as would be expected if the amino acid
residue at this position has the aforementioned block-
ing function. However, the level of increase is very
modest (approximately three-fold) compared to a vari-
ant in which the C-terminal domain was removed
completely by trypsin digest (50- to 100-fold). How-
ever, it should be noted that copper analysis revealed

that this mutant was very underloaded with copper
(only 1.2 equivalents per mole rather than the
expected 2). It could be reasonably expected that a
higher level of loading would allow much greater
levels of activity.
Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho
¨
ny-Meyer
2090 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS
The importance of Phe453 in pro-tyrosinase is also
indicated by the fact that we could not induce wild-
type pro-tyrosinase to form its active oxy complex, as
indicated by its absorbance spectrum, whereas the
F453A mutant, similar to the recombinant core tyrosi-
nase, readily formed this complex (Fig. S4). These
results suggest that the Phe453 residue is in the close
vicinity of the enzyme active site and plays some role
in oxygen binding.
Nonfunctional tyrosinase mutants
To further investigate the function of various other
amino acids in V. spinosum tyrosinase, we also con-
structed two further mutants. The importance of
Arg40 as a potential residue interacting with Tyr347
and Tyr348 was tested by changing the arginine to an
alanine. However, the mutation abolished the expres-
sion of recombinant protein completely (not shown),
which could indicate this residue is vital for protein
stability.
We also attemted to test whether Cys84 of the
V. spinosum tyrosinase has a similar role in forming an

alkane thiol bond, as has been shown for the sweet
potato catechol oxidase [8] or the N. crassa tyrosinase
enzyme [15]; therefore, this residue was mutated to a
serine in the pro-tyrosinase. Unlike in A. oryzae, where
a similar mutation resulted in a loss of activity but not
of expression [46], we found that this mutation resulted
in a complete loss of detectable protein. This suggests
that the residue is essential for correct folding and
expression of the enzyme. This appeared to contradict
the results obtained with the A. oryzae enzyme; how-
ever, it should be noted that this is a unique tyrosinase
that has a novel acid-induced self-activation mecha-
nism [47]. Furthermore, it has been shown to change
from a tetramer in the pro-form to a disulfide-linked
dimer in the mature form. Because the V. spinosum
pro-tyrosinase, its trypsinized form and the recombinat
core domain were all found to be monomeric, they are
probably not directly comparable to the A. oryzae
enzyme (Fig. S2).
Verrucomicrobium spinosum tyrosinase as an
alternative model bacterial enzyme
In summary, we present a system that allows the
expression of high levels of a novel bacterial tyrosi-
nase. This system has the advantage of an accessory
copper chaperone not needing to be expressed for
copper reconstitution because the protein can be
expressed in the apo-form and reconstituted after
purification. The expression and purification of the
apo-form prevents melanin formation during culture
growth, which greatly simplifies downstream process-

ing and improves protein yields. The resulting enzyme
preparations have been demonstrated to have high lev-
els of tyrosinase activities provided the inhibitory
C-terminal domain is removed either by proteolysis or
recombinant expression. The recombinant V. spinosum
tyrosinase constructs should prove useful for the
investigation of non-streptomyces type tyrosinases and
may also allow the determination of a crystal structure
of a tyrosinase in its low activity pro-form as well as
the solution of a structure that is not in complex with
a caddie protein.
Experimental procedures
Materials
Chemicals and proteins were purchased from Sigma-
Aldrich, molecular biology reagents from Fermentas GmbH
(Le Mont-sur-Lausanne, Switzerland) and oligonucleotides
from Microsynth AG (Balgach, Switzerland). Chromatogra-
phy resins and columns were purchased from GE Health-
care Europe GmbH (Bjo
¨
rkgatan, Sweden).
Molecular biology and molecular cloning
Verrucomicrobium spinosum (strain No. 4136) was obtained
from DSMZ GmbH (Braunschweig, Germany) and cul-
tured under the recommended conditions [48]. Primers Ver-
rucFP01 and VerrucRP01 were used to amplify the
tyrosinase gene (Pubmed Locus Tag VspiD_010100001190)
and were designed using the draft genome from TIGR
(Project ID: 10620). The full-length gene was then cloned
into the BamHI and HindIII sites of pUC18 and the

sequence verified using the Synergene Biotech GmbH
(Zurich, Switzerland) sequencing service. Mutants were
made using standard PCR techniques or QuikchangeÔ
(Stratagene, La Jolla, CA, USA) mutagenesis using the
primers listed in Table S1.
Protein expression
For protein expression, the full-length tyrosinase insert or
mutants thereof were sub-cloned into the EcoRI and
HindIII sites of the pQE60 vector (Qiagen AG, Hom-
brechtikon, Switzerland) using the VerrucRBSFP01 ⁄
VerrucRP01 or VerrucRBSFP02 ⁄ VerrucRP01 primer pairs.
The resulting plasmids (Table 1) were transformed into
E. coli strain DH5a. Constructs were tested for melanizing
activity by streaking transformed cells onto M9-agar
plates [19] containing 100 lm CuSO
4
,1mm isopropyl thio-
b-d-galactoside, 100 lgÆmL
)1
ampicillin, 1% glycerol and
0.5 mgÆmL
)1
(2.76 mm) l-tyrosine. The plates were then
M. Fairhead and L. Tho
¨
ny-Meyer Recombinant V. spinosum tyrosinase
FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2091
incubated at 37 °C overnight and visually checked the next
day for the formation of melanin.
For 1 L scale expression of the pro-tyrosinase and its

F453A mutant, a 30 mL overnight culture was grown from
a single transformant in LB [49] + 1% glucose + ampicil-
lin 100 lgÆmL
)1
. The overnight culture was used to inocu-
late (1 : 50) 2 · 500 mL of M9 + medium, containing: M9
salts, 1% peptone, 1% glycerol and 1% glucose, 100 lm
calcium (CaCl
2
), 2 mm magnesium (MgSO
4
), 100 lm thia-
mine and 100 lgÆmL
)1
ampicillin in 2 · 2 L Erlenmeyer
flasks. This culture was grown at 37 °C with shaking at
180 r.p.m. for 4–5 h, D
600
 0.5, then 1 mm isopropyl thio-
b-d-galactoside and 100 lgÆmL
)1
ampicillin was added and
growth continued for another 20 h.
Expression of the recombinant core domain of tyrosinase
was performed using modified autoinduction media [50].
A 30 mL overnight culture was grown from a single trans-
formant in LB + 1% glucose + ampicillin 100 lgÆmL
)1
.
The overnight culture was used to inoculate (1 : 50)

2 · 500 mL of auto induction media: 1% N-Z-amine, 0.5%
yeast extract, 25 mm Na
2
HPO
4
,25mm KH
2
PO
4
,50mm
NH
4
Cl, 5 mm Na
2
SO
4
, 1% glycerol, 0.4% lactose, 0.5%
glucose, 100 lm CaCl
2
,2mm MgSO
4
, 100 lm thiamine and
100 lgÆmL
)1
ampicillin in 2 · 2.5 L full baffle Tunair flasks
(Shelton Scientific, Shelton, CT, USA). This culture was
grown at 37 °C with shaking at 160 r.p.m. for 24 h.
Protein purification
Cells were harvested by centrifugation and washed in 0.1 m
Tris-HCl (pH 8). The washed cell pellet was resuspended

using 2 mL of 0.1 m Tris-HCl (pH 8) per gram wet weight
of cells, to which lysozyme was added to 1 mgÆmL
)1
. Cells
were incubated for 1 h on ice and then frozen at )80 °C.
Cells were then thawed and sonicated with a Branson soni-
fier cell disruptor (Branson Ultrasonics Corp., Danbury,
CT, USA), equipped with a 13 mm tip on 50% power
using five 20 s bursts. The sample was then centrifuged at
50 000 g for 30 min. To the soluble fraction, 0.6 g ⁄ mL of
NH
4
SO
4
was then added and the sample centrifuged at
50 000 g for 30 min. The resulting pellet was dissolved in
20 mL of 0.1 m Tris-HCl (pH 8) and dialysed against 5 L
of 10 mm Tris-HCl (pH 8) for 2 h, at which point the buf-
fer was exchanged and dialysis continued overnight. The
dialysed sample was then centrifuged at 50 000 g for
30 min. The desalted sample was then passed over a
160 mL bed volume Q-Sepharose fast flow column (GE
Healthcare Europe GmBH) and the unbound fraction con-
taining tyrosinase collected, running 10 mm Tris-HCl buffer
(pH 8). Tyrosinase containing fractions were then pooled
and concentrated to 5 mL and loaded onto Superdex 75
16 ⁄ 60 gel filtration column (GE Healthcare Europe
GmBH), 120 mL bed volume, running 10 mm Tris-
HCl + 0.1 m NaCl buffer (pH 8). Tyrosinase containing
fractions were then pooled concentrated to 10 mgÆmL

)1
and stored at –80 °C in 100 lL aliquots. All purification
steps were performed using an A
¨
KTA purifier 100 FPLC
(GE Healthcare Europe GmbH). The calculated extinction
coefficients at 280 nm were used to measure the concentra-
tion of the purified proteins (Table 1).
Size determination
For analytical gel filtration, a Superdex 75 16 ⁄ 60 column
was used, 120 mL bed volume, running 10 mm Tris-
HCl + 0.1 m NaCl buffer (pH 8). A calibration curve for
size determination was made using blue dextran (2 MDa)
and the proteins: horse heart cytochrome c (12.4 kDa),
horse heart myoglobin (17 kDa), bovine b-lactoglobulin
(35 kDa), ovalbumin (44.3 kDa) and bovine serum albumin
(67 kDa) (Fig. S2). The sizes of purified proteins was also
determined using the mass MS service of the ETH func-
tional genomics centre Zurich ( />Enzyme assay
Kinetic characterization of l-tyrosine and l-DOPA oxidation
was measured by dopachrome formation [51] at 475 nm using
a molar extinction coefficient of 3600 M
)1
Æcm
)1
at 25 °Cin
3 mL of 0.1 m potassium phosphate buffer (pH 6.8) using a
stirred Peltier assembly, with the spectra being monitored on
a Cary 50 bio UV ⁄ visible spectrophotometer (Varian Inc.,
Zug, Swizerland). Kinetic parameters were calculated using

prism
5 (GraphPad Software Inc., San Diego, CA, USA).
Bioinformatics
The molecular mass and theoretical extinction coefficient of
the various proteins were calculated using the protparam
tool available through the ExPasy server (-
asy.ch/tools/protparam.html) [52]. The signalP server was
used for signal peptide prediction ( />services/SignalP/) [53].
Copper reconstitution
Purified apo-tyrosinase was reconstituted with copper by
mixing an aliquot of protein ( 10 mg) with an equal vol-
ume of 10 mm Tris-HCl (pH 8), containing a three-fold
molar excess of CuSO
4
in a final volume of 1 mL. The sam-
ple was incubated on ice for 1 h and then dialysed twice
against 1 L of 10 mm Tris-HCl buffer (pH 8).
Copper analysis
The copper concentration of the protein samples was mea-
sured using a slight modification of the biquinoline method
[54]. Briefly 100 lLof10mgÆmL
)1
protein sample
was added to 0.2 mL of 0.1 m sodium phosphate
Recombinant V. spinosum tyrosinase M. Fairhead and L. Tho
¨
ny-Meyer
2092 FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS
buffer + 10 mm ascorbate (pH 6). To this, 0.7 mL of gla-
cial acetic acid containing 0.5 mgÆmL

)1
of 2,2-biquinoline
was added. The mixture was incubated for 10 min at room
temperature and A
546
was measured, using water as a refer-
ence. A standard curve using 0–165 lm CuCl
2
Æ2H
2
O was
also made and gave a calculated e for the copper biquino-
line complex of 5982 m
)1
Æcm
)1
.
Trypsinization
Trypsin digest of purified pro-tyrosinase was performed by
dissolving 20 lg of proteomics grade TPCK treated porcine
trypsin (Sigma-Aldrich) in 50 lLof1mm HCl and mixing
it with an aliquot of pro-tyrosinase ( 10 mg), final volume
1 mL in 0.1 m Tris-HCl buffer (pH 8). The sample was
then incubated at room temperature for up to 24 h.
Chemical denaturation of proteins
Unfolding experiments using GdnCl as a denaturant were
performed by diluting protein solutions in 10 mm Tris-HCl
buffer (pH 8) containing varying concentrations of GdnCl
(0–6 m); the final protein concentration in each sample was
0.1 mgÆmL

)1
, all samples were incubated for 24 h at room
temperature prior to fluorescence measurements. The fluo-
rescence of each sample was subsequently measured on a
Cary Eclipse spectrofluorimeter (Varian Inc.) at 25 °C, with
excitation at 285 nm and emission at 300–400 nm, and the
unfolding curve was calculated [55].
Acknowledgements
The authors wish to thank Linda Fahrni for technical
assistance and Dr Julian Ihssen and Dr Matthijs De
Geus for critically reading the manuscript.
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Supporting information
The following supplementary material is available:
Fig. S1. DNA and amino acid sequence of V. spinosum
tyrosinase.
Fig. S2. Calibration curve for size determination of

selected tyrosinases.
Fig. S3. Unfolding curves of selected tyrosinases.
Fig. S4. Absorbance spectra of selected tyrosinases.
Table S1. List of primers used in the present study.
This supplementary material can be found in the
online version of this article.
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should be addressed to the authors.
M. Fairhead and L. Tho
¨
ny-Meyer Recombinant V. spinosum tyrosinase
FEBS Journal 277 (2010) 2083–2095 ª EMPA, Swiss Federal Laboratories for Materials Testing and Research. Journal compilation ª 2010 FEBS 2095