A tyrosinase with an abnormally high tyrosine
hydroxylase/dopa oxidase ratio
Role of the seventh histidine and accessibility to the active site
Diana Herna
´
ndez-Romero
1
, Antonio Sanchez-Amat
1
and Francisco Solano
2
1 Department of Genetics and Microbiology, 2 Department of Biochemistry and Molecular Biology B, University of Murcia, Spain
Polyphenol oxidases (PPOs) are a broad group of cop-
per enzymes able to catalyze the oxidation of a great
variety of phenols by molecular oxygen [1]. Basically,
there are two main types of PPO, laccases and tyrosin-
ases, with significant differences at the polypeptidic cop-
per-binding sites [2] and the spectroscopic properties of
the metal ions [3,4]. Both enzymes are widely distributed
in nature. The active site of tyrosinases consists of a pair
of coupled copper ions called copper type-3. However,
blue laccases have up to four copper ions at the active
site of three different types, one type-1, one type-2 and
a couple of type-3. Tyrosinases catalyse the hydroxyla-
tion of monophenols to o-diphenols (cresolase or mono-
phenolase activity) and the subsequent oxidation of
o-diphenols to o-quinones (catechol oxidase or dipheno-
lase activity) [5,6] (Fig. 1). One of the most common
monophenolic substrates in a variety of organisms is
tyrosine, justifying the activity tyrosine hydroxylase
for monophenolase. The product of this hydroxylation

characteristics are discussed in relation to two other characteristics apart
from the six conserved histidines. One is the putative presence of a seventh
histidine which interacts with the carboxy group on the substrate and con-
trols the preference for carboxylated and decarboxylated substrates. The
second is the size of the residue isosteric with the aromatic F261 reported
in sweet potato catechol oxidase which acts as a gate to control accessibil-
ity to CuA at the active site.
Abbreviations
DO, dopa oxidase; dopachrome, 2-carboxy-2,3-dihydroindole-5,6-quinone; PPO, polyphenol oxidase; R3, a wild-type strain of R. solanacearum
spontaneously resistant to rifampicin; R3-0337
–
, R3 mutated in RSc0337 gene; R3-1501
–
, R3 mutated in RSc1501 gene; TH, tyrosine
hydroxylase.
FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 257
particular catechol to o-dopaquinone is also called dopa
oxidase (DO) activity. On the other hand, laccases oxid-
ize mainly p-diphenols and methoxy-substituted mono-
phenols to finally yield, respectively, p-quinones and
dimeric quinonic structures (Fig. 1) [7].
Tyrosinases are responsible for vertebrate cutaneous
pigmentation, browning of fruits and vegetables, and
morphogenesis and fruiting body formation in fungi.
All of these processes involve melanin formation. In
the bacterial kingdom there are some examples of well-
characterized tyrosinases. They were first described in
the genus Streptomyces [8,9], but the enzyme has also
been reported in other bacteria such as Sinorhizobium
meliloti [10] and Marinomonas mediterranea [11]. The

Different names are used for these activit-
ies, as shown in the Figure. One of the
most common monophenolic substrates is
tyrosine, and in that particular case the
activities are named tyrosine hydroxylase
(TH) and dopa oxidase (DO). (B) In this case,
the product of the catalysis, o-dopaquinone
is rapidly converted into dopachrome, the
colored product measured in the spectro-
photometric assay. (C) Laccases are differ-
ent PPOs with the capacity to oxidize
p-diphenols or methoxy-monophenols.
A novel tyrosinase from Rastonia solanacearum D. Herna
´
ndez-Romero et al.
258 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS
relevant features for addressing the molecular deter-
minants of bacterial pathogenicity to plants. It is a
soil-borne pathogen which naturally infects roots. It
exhibits a strong and tissue-specific tropism within the
host, invading and multiplying in the xylem vessels. In
addition, this b-proteobacterium has an unusually wide
host range. The genome of the strain GMI1000 isolated
from tomato has been sequenced [19]. It contains up to
four genes that putatively code for copper PPOs. We
have recently proved that at least three of these genes
are expressed and the corresponding protein products
show PPO activity, including two tyrosinase-like
enzymes and one laccase [17].
The monophenolase activity of tyrosinases is usually

much easier than hydroxylation of monophenols. The
noncatalyzed reaction rate for the atmospheric oxygen
oxidation of o-diphenols to o-quinones is several orders
of magnitude faster than that for monophenol
hydroxylation to o-diphenols. Pigment cell researchers
should be aware that stock solutions of l-dopa darken
spontaneously because of its oxidization, especially at
neutral or basic pH, but stock l-tyrosine solutions are
stable for long periods.
In this paper, we show that one of the two tyrosin-
ase-like PPOs produced by R. solanacearum displays
higher tyrosine hydroxylase (TH) than DO activity. To
our knowledge, this is the first tyrosinase with this very
interesting feature. Comparison of the amino acid
sequences at the active site with other tyrosinases and
catechol oxidases allows us to propose correlations
between key residues in the catalytic patterns of these
enzymes and whether they act as true tyrosinases
(monophenolases plus o-diphenolases) or only o-diphe-
nolases.
Results
Genes encoding putative tyrosinases
in R. solanacearum
After genome sequencing of R. solanacearum, two
genes that putatively code for tyrosinase-like enzymes
were detected by a blast search [19]. They were named
catechol oxidase (gene RSc0337, protein NP_518458)
and tyrosinase (gene RSc1501, NP_519622).
When we submitted both sequences to a hierarchical
multiple sequence alignment [25], two sets of proteins

FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 259
Table 1. Alignment of sequences at the CuA and CuB binding sites of PPOs from R. solanacearum, NP_518458 (gene RSc0337) and NP_519622 (gene RSc1501) with the proteins show-
ing highest sequence similarity (scores higher than 100 and e values lower than 10
)21
in all cases). PPOs from I. batata and A. bisporus have been included as important model PPOs. The
six H residues directly involved in copper binding are marked in shadow background, and the other proposed positions related to the monophenolase vs. o-diphenolase differences are in
bold and higher size. Consensus shows the concordance with the conserved positions for tyrosinases [6].
A novel tyrosinase from Rastonia solanacearum D. Herna
´
ndez-Romero et al.
260 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS
logous recombination. Briefly, the gene RSc1501 was
amplified by PCR from genomic DNA of a spontaneous
Rif
R
R. solanacearum wild-type GMI1000 strain which
we called R3. The PCR product with a size of  1.6 kb
was digested with BamHI to obtain a fragment between
the two copper-binding site coding regions and ligated
to pBlueScript pKSII(+) with T4 DNA ligase (Invitro-
gen, San Diego, CA, USA). The ligation mixture was
transformed in Escherichia coli DH5a, and transform-
ants selected for ampicillin resistance. The plasmid
obtained (pBRI15) was digested with EcoRI and SacI,
and the internal RSc1501 gene fragment subcloned in
the pFSVK plasmid. The resulting plasmid (pCN15)
was transformed in E. coli S17-1 (kpir), and transform-
ants selected for kanamycin resistance. The plasmid in
this strain was mobilized into spontaneous Rif
R

was 0.05%, the same as optimal TH conditions [17].
Furthermore, when these activities were determined in
cellular extracts of the mutant strains generated and
compared with the wild-type strain, we found that
each activity was lost in extracts of different mutants.
Mutation of the RSc0337 gene resulted in loss of
almost all TH activity, whereas mutation of the
RSc1501 gene resulted in loss of most of the DO activ-
ity, indicating a correspondence between both activities
and the proteins encoded by the respective mutated
genes, which was opposite to that expected from the
blast homologies and designated names (Fig. 2).
Moreover, the TH activity in both mutants showed
a very different dependence on l-dopa as cofactor to
eliminate the characteristic lag period of tyrosinases
[8,20,21]. Figure 3 shows the rate of TH activity as a
function of the concentration of l-dopa cofactor added
to the assay mixture. R3-1501
–
extracts have a high
TH activity, almost independent of the addition of
l-dopa cofactor, and the lag period before reaching
the maximal reaction rate without this addition is
short ( 40–60 s under standard conditions). The TH
activity of R3-0337
–
extracts is quite low and needs to
Fig. 2. TH and DO activities in extracts of wild-type R3 R. solana-
cearum and two mutant strains with mutations in the PPO genes
RSc0337 and RSc1501. TH activity was determined at pH 5 and

Purification of two enzymes with different
affinities for monophenols and o-diphenols
Supernatants of bacterial crude extracts obtained from
R3 wild-type and mutant strains were submitted
to enzyme purification. These supernatants, routinely
 30 mL, were first concentrated 5–6 times using ultra-
filtration membranes (Millipore; cut-off 10 kDa) and
applied to CM-Sephadex A-50 chromatography in
0.05 m sodium phosphate buffer, pH 7, according to
the basic pI predicted from their amino-acid sequence.
After elution of unbound proteins, the ionic strength
was increased with a salt gradient of NaCl up to 1.5 m
to elute proteins bound to the anionic gel. Fractions of
1.9 mL were collected, the protein content was monit-
ored (A
280
), and TH and DO activities were assayed
under the respective optimal conditions.
The purification profiles of bacterial extracts from
wild-type (R3-wt), mutant strain R3-1501
–
affected in
the NP_519622 protein and mutant strain R3-0337
–
affected in the NP_518458 protein are shown in Fig. 4,
and a summary of the purification is shown at Table 2.
Apart from a small amount of DO activity found in the
large peak of unbound proteins eluted before applica-
tion of the salt gradient, two PPOs were eluted in the
wild-type strain at high salt concentration,  0.9 and

respective specific activities ensure minimum turnover
numbers of 750 and 1550 min
)1
for the TH activity
of the monophenolase and the DO activity of the
o-diphenolase, respectively.
Affinity for carboxylated and decarboxylated
phenolic substrates
To explore the affinity of the active site of the two
PPOs for phenolic substrates and possible correlations
between the structural requirements for interaction and
Fig. 4. Purification profiles in CM-Sephadex chromatography of cel-
lular extracts from wild-type and mutant strains. After elution of all
unretained proteins, a linear gradient of NaCl up to 1.5
M in the
same buffer was applied to the column. A
280
, TH and DO stand,
respectively, for the profile of UV absorbance (total protein) and
enzymatic PPO activities. (A) Wild-type R3 strain; (B) R3-1501
–
;
(C) R3-0337
–
.
A novel tyrosinase from Rastonia solanacearum D. Herna
´
ndez-Romero et al.
262 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS
the differences between the two PPOs, the kinetics

cases, TH activity was determined at pH 5 with 2 m
ML-Tyr and 0.05% SDS, and DO activity at pH 7 with 2 mML-Dopa and 0.02% SDS. In
column A, 49 and 9 are, respectively, the amounts of protein (lg) in the TH and DO activity peaks. Yields were calculated with the values in
parentheses, which are the three most active fractions from the purification peaks pooled, but maximal purification (n-fold) was calculated
from the most active fraction. wt, Wild-type.
Crude Ultrafiltrate Purified fraction
Column A: wt, R3 extract (contains both enzymes)
Proteins (lg) 79200 48050 49 & 9 (2 peaks)
Total activity of TH (mU) 3248 1505 493 (1097)
Total activity of DO (mU) 3540 1685 278 (869)
Specific activity of TH (mUÆmg
)1
) 41.0 31.3 10056
Specific activity of DO (mUÆmg
)1
) 44.7 35.1 30888
Purification (n-fold) ⁄ yield TH (%) 1 ⁄ 100 0.8 ⁄ 46.3 245 ⁄ 34
Purification (n-fold) ⁄ yield DO (%) 1 ⁄ 100 0.8 ⁄ 47.6 691 ⁄ 25
Column B: R3-1501
–
(contains NP_518458)
Proteins (lg) 16000 9200 12
Activity of TH (mU) 1997 1140 210 (579)
Specific activity of TH (mUÆmg
)1
) 124.8 123.9 17500
Purification (n-fold) ⁄ yield TH (%) 1 ⁄ 100 1 ⁄ 57.1 122 ⁄ 29
Column C: R3-0337
–
(contains NP_519622)

the
L-tyrosine ⁄ tyramine and L-dopa ⁄ dopamine pairs. Kinetic parame-
ters are summarized in Table 3.
D. Herna
´
ndez-Romero et al. A novel tyrosinase from Rastonia solanacearum
FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 263
a marked loss of affinity for the substrate (the K
m
increased to  10 mm; data not shown) and low reac-
tion rates.
Concerning diphenolase activity, the enzyme
NP_518458 was a poor catalyst, but again it preferred
the carboxylated o-diphenol (l-dopa) over its decar-
boxylated counterpart, dopamine. On the other hand,
the NP_519622 protein showed very efficient dipheno-
lase activity, particularly with dopamine. Activities
with these o-diphenol substrates were higher than
1000 mUÆmg
)1
(Table 3), although the affinity was not
very high. To summarize, protein encoded by RSc0337
is an efficient monophenolase, especially with carboxyl-
ated monophenols, but the protein encoded by
RSc1501 is an efficient diphenolase, especially with
decarboxylated o-diphenols.
Dopa accumulation in the TH reaction catalysed
by the NP_518458 protein
Figure 7A shows the stoichiometric formation of
2-carboxy-2,3-dihydroindole-5,6-qu inone (l-dopachrome)

Enzyme Activity V
max
(mUÆmg
)1
) K
m
(mM)
Cat. efficiency
(mUÆmg.mM
)1
)
NP_518458 TH 254.4 1.32 192.7
TaH 59.2 2.54 23.3
NP_519622 TH 106.7 0.94 113.5
TaH 198.9 1.18 168.6
NP_518458 DO 46.8 2.87 16.3
DaO 8.2 0.95 8.7
NP_519622 DO 1264.0 3.53 358.1
DaO 3075.0 3.87 794.6
A
B
Fig. 7. (A) Time-course accumulation of L-dopachrome and L-dopa
during the TH activity of NP_518458.
L-dopachrome (n) is formed
directly and monitored continuously, but
L-dopa (m) was titrated by
addition of excess sodium periodate at several fixed times of reac-
tion. (B) Catalytic cycles for the monophenolase (up, clockwise) and
o-diphenolase (down, anticlockwise) activities. MF, Monophenol;
DF, o-diphenol; Q, o-quinone; T, tyrosinase. T has three different

account that melanogenesis is related to virulence of
the infective micro-organism, but it is also related to
defensive roles in the infected cell, so that the place
and time of triggering of melanogenesis must be key to
the success of one of these two opposite processes. In
turn, a melanogenic complex has been described in
mammals between tyrosinase and tyrosinase related
protein 1 [28]. The latter can behave as an o-dipheno-
lase-like protein but also as a stabilizing protein for
true tyrosinase [29]. In R. solanacearum, NP_518458
would mainly catalyse the rate-limiting step, monophe-
nol hydroxylation, and NP_519622 would catalyse the
second step, oxidation of o-diphenol to o-quinone, or
alternatively a stabilization of the former enzyme.
Studies on possible interactions between the PPOs are
underway in our laboratory. On the other hand, envi-
ronmental conditions, for instance acidic or neutral
environmental pH, may also affect the expression of
the most appropriate enzyme.
Apart from the physiological roles and environmen-
tal advantages of having several PPOs in the same
organism, we have found that the RSc0337 gene codes
for an enzyme with high TH activity and lower DO
activity, with optimum assay conditions at pH 5,
whereas the RSc1501 gene codes for an enzyme that
efficiently oxidizes l-dopa, although it also shows low
activity with l-tyrosine, as revealed by the residual
TH activity detected in the R3-0337
–
mutant. Its opti-

l-dopa cofactor to reach maximal tyrosine hydroxylase
activity. To our knowledge, this feature is not found in
any other reported tyrosinase, from Streptomyces to
mammals. The turnover number of tyrosinases for DO
is about 100 times higher than for tyrosine hydroxyla-
tion [21]. In this regard, fungal and bacterial tyrosinases
are very similar, showing a higher k
cat
and activity
with o-diphenols than with monophenols [8]. More-
over, the TH ⁄ DO ratio is almost zero in plant catechol
oxidases lacking monophenolase activity. In general,
o-diphenols bind more rapidly to oxy-tyrosinase than
monophenols [4,30]. However, this tyrosinase from
Fig. 8. Stability of proteins NP_518458 (TH activity) and NP_519622
(DO activity) in phosphate buffer, pH 7. Both purified PPOs were
submitted to heat (60 °C) or high SDS concentration (0.5%).
D. Herna
´
ndez-Romero et al. A novel tyrosinase from Rastonia solanacearum
FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 265
R. solanacearum has the opposite kinetic properties. In
contrast with all other tyrosinases, the TH ⁄ DO data
summarized in Table 4 clearly show that the monophe-
nol is the preferred substrate.
Tyrosinases catalyse monophenolase hydroxylation
and ⁄ or o-diphenolase oxidation as shown in Fig. 7B.
Binding of monophenols to resting met-tyrosinase
results in the inactive dead-end complex, but binding
of o-diphenols leads the enzyme to the oxy-tyrosinase

exploring this is comparison of crystal structure data.
The only data so far available are for sweet potato
(Ipomoea batata) catechol oxidase [26]. The catalytic
copper center is accommodated in a central four-helix
bundle located in a hydrophobic pocket, with the six
histidines bound to the copper pair. This particular
enzyme behaves as a catechol oxidase as it does not
show monophenolase activity, and the o-diphenol
binds to CuB [4,32].
The most likely explanation for the lack of mono-
phenolase activity of this PPO is related to the position
of the bulky aromatic residue F261. In sweet potato
o-diphenolase, F261 blocks access to CuA [4,26]. This
aromatic residue acts as a gate, controlling the accessi-
bility of phenolic substrates to the hydrophobic pocket
where the dinuclear copper center is found. In addition,
van der Waals interactions between this aromatic resi-
due lining the hydrophobic cavity and the aromatic
ring of phenolic substrates help to determine the affin-
ity of substrates for the enzyme. In wild-type and
mutated mouse tyrosinase, it was proposed that the
absence of this aromatic residue at the equivalent posi-
tion may be the reason why it shows monophenolase
activity, assuming that residue controls the access of
monophenols to CuA [31]. Although monophenols and
o-diphenols could access CuB, F261 may block the
re-orientation of monophenols toward CuA that is nee-
ded for its hydroxylation once is bound to CuB [32]. It
is very unlikely that minor details can be universally
extrapolated to all tyrosinases and catechol oxidases

Preference substrate ⁄
preferred name
NP_518458 pH 5 5.4 7.2 Carboxylated
monophenols ⁄
tyrosinase
NP_519622 pH 7 0.08 0.06 Decarboxylated
o-diphenols ⁄
catechol oxidase
A novel tyrosinase from Rastonia solanacearum D. Herna
´
ndez-Romero et al.
266 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS
it is not the only factor. Other residues must be
involved in the mechanism of catalysis. It is also known
that both PPO types, tyrosinases and catechol oxidases,
show very different behavior with carboxylated and
decarboxylated substrates. According to Fig. 6 and
the summary in Table 3, tyrosinase shows more affinity
for and catalytic efficiency with carboxylated sub-
strates, l-tyrosine vs. tyramine and l-dopa vs. dopam-
ine. The situation is the opposite for catechol oxidase.
The difference between carboxylated and decarboxyl-
ated substrates is related to the difference between
monophenols and o-diphenols as favored substrate,
and also related to the presence or absence of a seventh
histidine, adjacent to the sixth histidine involved in
copper binding. Preceding the sixth histidine
(H
3B
according to the nomenclature used in [6]), at the

residue, thus its requirement is very doubtful. Rather
than a proton acceptor contributing to catalysis, this
E seems to be an obstacle to TH activity.
On the other hand, having monophenolase activity
does not directly mean that this is the favored activity.
The formerly discussed three positions confirm that
NP_518458 can act on carboxylated monophenols, but
they are not enough to account for its high TH ⁄ DO
ratio. This special feature must be due to a unique resi-
due(s) of this tyrosinase. With regard to this, the pair
of MM residues found just before the double HH at the
CuB-binding site is particularly interesting. It has
recently been reported that, in copper protein type-1,
axial methionines in positions close to the copper ion
greatly affect the redox potential and catalytic efficiency
[33]. Extrapolation to copper type-3 is an appealing
possibility. Site-directed mutagenesis needs to be per-
formed to clarify which factors are actually responsible
for the catalytic properties of this PPO, and experi-
ments on this are being carried out in our laboratory.
Apart from the interest of this novel tyrosinase as a
model for the mechanism of catalytic cycles, and its
His 81
Cys 91
Ala 241
His 93
His 153
His 163
His 172
His 283

applications that need a high monophenolase activity
accompanied by low o-diphenolase activity. In addi-
tion, this enzyme is quite resistant to temperature and
chaotropic agents in comparison with tyrosinases from
other sources. Streptomyces glaucescens has a very
labile enzyme, showing a half-life at 60 °Cof 5 min
[8], but tyrosinase from R. solanacearum is quite resist-
ant to that temperature and SDS, although there are
more resistant tyrosinases, such as the enzyme from
Thermomicrobium roseum, which is almost unaffected
by this temperature [34]. Further studies are necessary
to explore the possible biotechnological applications of
these enzymes.
Experimental procedures
Cell culture
R. solanacearum was grown in basal saline medium con-
taining 15 mm (NH
4
)
2
SO
4
, 0.8 mm MgCl
2
,2lm FeSO
4
,
0.2 mm CaCl
2
,8lm Na

280
was used to follow the protein
elution profile in chromatography purification columns.
Enzymatic determinations
TH and DO activities were determined by monitoring,
respectively, the oxidation of 2 mml-tyrosine or l-dopa to
l-dopachrome at 475 nm (Fig. 1B; e ¼ 3700 m
)1
Æcm
)1
), in
0.1 m sodium phosphate buffer. The pH was 5.0 or 7.0
according to the activity and PPO enzyme assayed due to
the different optimal conditions displayed by the activities
of this micro-organism [17]. Moreover, 0.05% SDS was
added for the standard TH assay, and 0.02% SDS was
added for DO activity. For dopa titration, 50 lL10mm
sodium periodate was added, and the increase in A
475
immediately determined. A small concentration of l-dopa
was occasionally added as cofactor for TH activity when
appropriate (detailed in results). Tyramine hydroxylase and
dopamine oxidase were monitored in the same way, but
dopaminochrome was determined (e ¼ 3100 m
)1
Æcm
)1
). In
all cases, one unit was defined as the amount of enzyme
that catalyses the appearance of 1 lmol dopachrome ⁄

BIO2004-4803 from CICYT, Spain. D.H.R. was been
supported by a financial grant associated with project
BIO2001-0140. Special thanks go to Professor Boucher
for supplying us with the sequenced strain.
A novel tyrosinase from Rastonia solanacearum D. Herna
´
ndez-Romero et al.
268 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS
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