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-

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

[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

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

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

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

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

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

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

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-

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

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

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

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-

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

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-

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

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

)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

)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

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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