Tài liệu Báo cáo khoa học: A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio Role of the seventh histidine and accessibility to the active site docx

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

is an o-diphenol, dopa, so that the oxidation of this
Keywords
catechol oxidase; copper enzymes;
monophenolase; phenol oxidase; tyrosinase
Correspondence
F. Solano, Department of Biochemistry and
Molecular Biology B, School of Medicine,
University of Murcia, Murcia 30100, Spain
Fax: +34 9683 64150
Tel: +34 9683 67194
E-mail address:
URL: www.um.es/bbmbi
(Received 25 July 2005, revised 6 October
2005, accepted 27 October 2005)
doi:10.1111/j.1742-4658.2005.05038.x
The sequencing of the genome of Ralstonia solanacearum [Salanoubat M,
Genin S, Artiguenave F, et al. (2002) Nature 415, 497–502] revealed several
genes that putatively code for polyphenol oxidases (PPOs). This soil-borne
pathogenic bacterium withers a wide range of plants. We detected the
expression of two PPO genes (accession numbers NP_518458 and
NP_519622) with high similarity to tyrosinases, both containing the six
conserved histidines required to bind the pair of type-3 copper ions at the
active site. Generation of null mutants in those genes by homologous
recombination mutagenesis and protein purification allowed us to correlate
each gene with its enzymatic activity. In contrast with all tyrosinases so
far studied, the enzyme NP_518458 shows higher monophenolase than
o-diphenolase activity and its initial activity does not depend on the pres-
ence of l-dopa cofactor. On the other hand, protein NP_519622 is an
enzyme with a clear preference to oxidize o-diphenols and only residual
monophenolase activity, behaving as a catechol oxidase. These catalytic

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

latter marine bacterium was the first prokaryote des-
cribed that expresses two different PPOs. One of them
is a soluble tyrosinase clearly involved in melanin
synthesis [11], and the second is a membrane-bound
laccase [12,13] with residual tyrosinase activity; its
physiological role is uncertain. In fact, the role and
physiological advantages of the coexistence of several
PPOs in the same micro-organism remain unknown.
The synthesis of melanin in micro-organisms has
been related to pathogenesis and virulence [14]. Melani-
zation of the infectious cell seems to offer an advant-
age, as microbial melanin could protect the pathogen
against the host cell [15], although melanization in the
host cells is also proposed to be part of the defense sys-
tem against wounding and infection by the pathogen
[16]. Thus, the timing and location of melanization
seems to be essential for the prevalence of one of these
two opposing processes. In any case, many of the bac-
teria that express PPO activities are strains that interact
with plants such as Rhizobium meliloti [10], Ralstonia
solanacearum [17] and the marine epiphyte Microbulbi-
fer degradans [18]. R. solanacearum has unique and
A
B
C
Fig. 1. PPO activities. These copper
enzymes show a wide range of action on
phenolic compounds. (A) Tyrosinases show
two activities, the hydroxylation of mono-
phenols and the oxidation of o-diphenols.

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

coupled to the o-diphenolase activity. In fact, it has
been proposed that tyrosinase binds monophenols
at the active site to directly oxidize these substrates to
o-quinones, so that the two activities cannot be separ-
ated [20]. In spite of this, o-diphenolase activity can be
determined by just using o-diphenols as the initial sub-
strate of tyrosinases.
Tyrosinases show a much higher specific activity for
oxidation of o-diphenols (o-diphenolase activity) than
for hydroxylation of monophenols (monophenolase or
cresolase activity) [5,21]. Furthermore, it is quite
common in plants to find PPOs that act exclusively as
o-diphenolases, with none or a very residual mono-
phenolase activity [16]. In animals, as well as a true
tyrosinase, there is another protein called Trp1, which
can be considered an o-diphenolase because it shows
low oxidase activity with two o-diphenols, dopa and
5,6-dihydroxyindole-2-carboxylic acid [22,23]. This is
the main reason why classical enzymology classifies the
same family of proteins with the pair of type-3 cop-
per ions in tyrosinases (monophenol l-dopa-oxygen
oxidoreductase, EC 1.14.18.1) and catechol oxidases
(o-diphenol–oxygen oxidoreductase, EC 1.10.3.1), but
the differentiation between these two types of enzyme
is not clear [4]. Looking at the sequences of the two
enzymes, both show absolute conservation of the histi-
dine residues of the CuA and CuB binding regions and
the same Prosite signatures [2,4,6].
The low or zero monophenolase ⁄ o-diphenolase ratio
is understandable. Chemical oxidation of o-diphenols is

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

showing highest sequence similarity were obtained [17].
Interestingly, these sets did not overlap. The protein
NP_518458 was found to be similar to several plant
catechol oxidases and a few bacterial proteins
(Table 1). Catechol oxidase from sweet potato (Ipo-
moea batatas) was not in the top five highest scoring
proteins, but it is included in the table because it is the
only enzyme of this family that has an available crystal
structure [4,26]. The similarity to plant catechol oxid-
ases supports the initial naming of this protein [19].
On the other hand, the proteins with highest sequence
similarity to NP_519622 were several Streptomyces
tyrosinases (Table 1). This therefore justifies the nam-
ing of this enzyme as tyrosinase. Mushroom tyrosinase
is included in Table 1 because it is the most commonly
used tyrosinase in model studies. It is important to
note that the most characteristic signatures in the
sequences are present in both proteins, tyrosinases and
catechol oxidases; these include the six histidine resi-
dues involved in the binding of a pair of copper ions
and other conserved residues [6]. However, so far it is
not possible to predict from this signature the enzy-
matic activity that a protein will actually display.
Isolation of R. solanacearum mutants affected
in tyrosinase-like activities
Strains with mutations in the two genes coding for
tyrosinase-like activities were constructed by homo-
D. Herna
´
ndez-Romero et al. A novel tyrosinase from Rastonia solanacearum

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

R3
by conjugation [17]. RSc1501 gene disruption in the
transconjugants was confirmed by appropriate PCR
and product analysis (data not shown). One strain,
R3-1501
–
, was selected for further assays.
To obtain mutants affected in the RSc0337 gene, an
internal fragment of 300 bp between the two copper-
binding sites from this gene was amplified using the
appropriate forward and reverse primers. Then the
product was cloned in the pFSVK plasmid using
the NcoI and SacI restriction sites. The resulting plas-
mid pCN337 was transformed in E. coli and mobilized
into R3 as described above for the RSc1501 gene.
RSc0337 disruption was also confirmed in the trans-
conjugants by PCR [17], and one strain, R3-337
–
, was
selected for further studies.
PPO activity in R. solanacearum and mutants
affected in genes coding for these proteins
R. solanacearum showed monophenolase and o-dipheno-
lase activities, represented by TH and DO, respectively.
The conditions for the PPO enzymatic assays differed
with regard to pH and SDS concentration. TH activity
was higher at pH 5 and 0.05% SDS, but DO showed a
sharp peak at 0.02% SDS and pH 7. In fact, the rate
of oxidation of l-dopa was much lower at pH 5, but
under these conditions the optimal SDS concentration

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

0.05% SDS, and DO activity at pH 7 and 0.02% SDS.
Fig. 3. Dependence of the TH activity of extracts of R3 and the
two mutant strains on the presence of cofactor,
L-dopa. (n) R3; (d)
R3-0337
–
which only expresses the protein NP_519622; (m)
R3-1501
–
which only expresses the protein NP_518458. Lag peri-
ods in the absence of
L-dopa were, respectively, 58, 286 and 42 s.
All measurements represent the estimation of net
L-dopachrome
formed, after subtraction of the blanks in the absence of the sub-
strate,
L-tyrosine.
D. Herna
´
ndez-Romero et al. A novel tyrosinase from Rastonia solanacearum
FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 261
be activated by the addition of l-dopa. Its lag period
in the absence of l-dopa is  5 min. R3 wild-type
extracts behave much more like R3-1501
–
than R3-
0337
–
. This pattern agrees with the presence of two
different enzymes with overlapping activities.

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

1.05 m NaCl, respectively. The first one had high TH
activity, although it also had detectable DO activity
under the optimal conditions for this activity (0.02%
SDS, pH 7). The second one displayed only DO activity
under these conditions. Interestingly, the first peak but
not the second one was found in the extracts of
R3-1501
–
, and the opposite was observed in extracts of
R3-0337
–
mutant. This behavior clearly suggests that
these peaks are due to different enzymes, and that the
TH activity is due to the NP_518458 protein, whereas
the DO activity is mostly due to the NP_519662 protein.
As these proteins were preliminarily named catechol
oxidase and tyrosinase, respectively, this activity profile
strongly indicates that the names should be exchanged.
The main stages of the purification process for the
three extracts are summarized in Table 2. The initial
total amounts of protein are not the same because we
started purification from different amounts of material.
During the purification process, we obtained 245-fold
and 691-fold purification for the two wild-type PPOs,
and yields of  30%. These purification factors were
not so high when we used the mutant extracts as start-
ing material. The purified peaks of the two PPOs
showed purities greater than 90%, as judged by
SDS ⁄ PAGE, and apparent molecular masses of the
active enzymes of  35 and 50 kDa (Fig. 5). The

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

parameters of carboxylated ⁄ decarboxylated substrates
were calculated. We used the couples l-tyrosine ⁄ tyram-
ine for the monophenolase activity and l-dopa ⁄ dop-
amine for the diphenolase activity. Standard activities
under optimal conditions are shown in Fig. 6, and val-
ues for V
max
, K
m
and catalytic efficiencies in Table 3.
Concerning monophenolase activity, the enzyme
NP_518458 greatly preferred l-tyrosine to tyramine. It
showed higher V
max
and lower K
m
for the carboxylated
monophenol, which can be more clearly appreciated if
the catalytic efficiency (V
max
⁄ K
m
) is calculated. At pH
7 the affinity for these substrates was slightly lower
(data not shown). On the other hand, the enzyme
NP_519662 did not show preference for l-tyrosine. In
fact, this enzyme was a little bit more efficient in
tyramine hydroxylation. It was almost completely
unable to hydroxylate monophenols at pH 5, showing
Table 2. Purification of tyrosinase and catechol oxidase (proteins NP_518458 and NP_519622, respectively) from R. solanacearum. In all

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)

Proteins (lg) 40800 10100 8
Activity of DO (mU) 957 605 84 (475)
Specific activity of DO (mUÆmg
)1
) 23.5 60 10500
Purification (n-fold) ⁄ yield DO (%) 1 ⁄ 100 2.5 ⁄ 63 447 ⁄ 50
12
kDa
90
46
35
20
Fig. 5. SDS ⁄ PAGE of the most pure PPO fractions from Fig. 3A. 1,
First peak (elution volume, V
e
¼ 110 mL); 2, second peak (V
e
¼
123 mL). All the peaks showed purities of at least 90%. Similar sin-
gle bands were obtained with peaks obtained from R3-1501
–
and
R3-0337
–
.
Fig. 6. Comparison of the monophenolase ⁄ o-diphenolase activities
of extracts [mUÆ(mg protein)
)1
] from wild-type and mutated R. sol-
anacearum strains to carboxylated ⁄ decarboxylated substrates using

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)

and l-dopa during the time course of tyrosine
hydroxylation. The l-dopa accumulated by the sponta-
neous disproportion of dopaquinone can be titrated at
different periods of time by addition of sodium perio-
date. According to the high preference of the enzyme
encoded by the RSc0337 gene for the monophenols
and the general mechanism for the reaction of tyrosin-
ases (Fig. 7B), it can be seen that dopa is not con-
sumed by the enzyme through the o-diphenolase cycle,
as it is not a competitor with the monophenolase
cycle.
Stability of PPOs
The stabilities of both enzymes, monophenolase
NP_518458 and o-diphenolase NP_519662, were stud-
ied by heating to 60 °C and exposure to a relatively
high concentration (0.5%) of the chaotropic and dena-
turing agent SDS. Note that the concentration is at
least 10 times higher than the SDS used for optimal
assay conditions (Fig. 8). It can be observed that the
first PPO is very stable to both treatments, but the
second one is labile.
Discussion
We have found two different genes in R. solanacea-
rum coding for putative PPO proteins that contain
the typical signatures of tyrosinases, including the
CuA and CuB binding sites to ligand the copper
Table 3. Kinetic parameters for the two PPOs. The enzymes were
obtained from extracts of R. solanacearum strains mutated in the
gene encoding the alternative one. DaO, Dopamine oxidase; TaH,
tyramine hydroxylase.

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

forms during the cycles: met, resting tyrosinase with Cu(II); oxy,
oxygenated form with peroxide bound to Cu(II); deoxy, reduced
Cu(I) transient form with high affinity for oxygen. The efficiency for
both cycles depends basically on the affinity of oxyT for the mono-
phenol or o-diphenol. The enzymatic product, o-quinone, undergoes
a very fast spontaneous disprorportion to regenerated o-diphenol
and the ‘chrome’ (see Fig. 1B). Dopa can be chemically oxidized
very rapidly to dopachrome by sodium periodate.
A novel tyrosinase from Rastonia solanacearum D. Herna
´
ndez-Romero et al.
264 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS
type-3 pair [2,6]. In principle, it is unclear what phy-
siological advantages there are for bacteria to express
two proteins so similar in terms of enzymatic activity.
However, this situation has been found previously in
other bacteria. Genome sequencing of Streptomyces
avermitilis also revealed the presence of two tyrosin-
ase-like enzymes, although it was suggested that one
of those genes is not expressed, or shows a very low
level of transcription [27]. In addition, we have repor-
ted the existence and expression of a multipotent lac-
case and a tyrosinase in Marinomonas mediterranea
[11,13].
We have now found in R. solanacearum that the two
tyrosinase-like genes and the laccase-like gene are
indeed expressed [17]. One attractive advantage to hav-
ing more than one PPO is that these proteins may
interact with each other to form a stable and very effi-
cient melanogenic complex. It should be taken into

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-

mal activity is at pH 7. These preferred activities of
the two PPOs of R. solanacearum are opposite to the
names assigned to them when the genome of this bac-
terium was sequenced and the function of these con-
ceptual proteins was proposed [19]. On the basis of
blast homology, the NP_518458 protein from the
RSc0337 gene was named catechol oxidase, and the
NP_519622 protein encoded by the gene RSc1501 was
named tyrosinase. This was logical according to the
mathematical algorithm used for the blast search.
Score and e values depend on several factors, but
mostly the total length of the sequence used for the
blast. The shorter sequence (412 amino acids), coming
from the RSc1501 gene, more closely matches the
short sequences (Table 1), which are tyrosinases from
Streptomyces species [2], and these homologies led to
this enzyme being designated a putative tyrosinase.
The long sequence (496 amino acids), coming from the
RSc0337 gene, more closely matches long bacterial
tyrosinases and a series of plant catechol oxidases,
which are also long. This led to the designation of this
protein as a putative catechol oxidase. It is clear that
matching the whole sequence is not a good way of dis-
tinguishing tyrosinases from catechol oxidases.
Having clearly established that protein NP_518458
is a tyrosinase (monophenolase) rather than a catechol
oxidase (o-diphenolase), we observed that it is a very
unusual tyrosinase as it is a more efficient monopheno-
lase than o-diphenolase and its TH ⁄ DO ratio is clearly
higher than 1. In the same way, it does not need

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

form, the active species for both monophenolase and
o-diphenolase activity [5,30–32]. About 85% of resting
mushroom tyrosinase is found in the met form and
15% in the oxy form, so that the o-diphenol formed
by this 15% is enough to recruit the enzyme to the cat-
alytic cycle after a short time, showing the characteris-
tic lag period of tyrosinases before reaching maximal
reaction rate [5,7,30,31]. Note that the product of the
reaction, dopachrome, is chemically formed by a redox
disproportion from the true enzymatic product o-qui-
none (Fig. 1). R. solanacearum tyrosinase seems to be
almost completely in the oxy form, as judged by the
absence of lag period in the absence of l-dopa cofac-
tor. This indicates that the dead-end inactive complex
(Fig. 7B) is not formed in this particular enzyme.
Titration of the amount of l-dopa generated during
its TH activity with sodium periodate shows that this
o-diphenol is stoichiometrically accumulated with
dopachrome (Fig. 7A), but this is not so using mush-
room tyrosinase (data not shown). These data confirm
the great preference of oxy-tyrosinase for monophen-
ols, so that the DO activity is not competing with TH
during the course of the reaction, and the chemically
generated l-dopa is not consumed.
The structural difference between catechol oxidases
and tyrosinases has not yet been explained. Concerning
the crucial regions for catalytic activity and substrate
affinity, the six copper-binding histidines of the two
PPOs do not show any differences (Table 1), but some
distinctions must exist. The most reliable way of

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

from any source, but there is no doubt that this factor
is important for accessibility to (or involvement of)
CuA in the PPO active site in order for it to display
monophenolase and o-diphenolase activity or just the
latter activity. For instance, all catechol oxidases from
tomato, potato and beans have the aromatic residue at
the equivalent position (Table 1). However, Streptomy-
ces tyrosinases usually have the smallest residue, G,
there. In octopus hemocyanin, L2830 occupies the posi-
tion of F261, and this may be responsible for the weak
o-diphenolase activity detected in this protein, as an L
residue blocks CuA less effectively than F.
Our results on the two PPOs found in R. solanacea-
rum are totally in agreement with this steric hin-
drance (Table 1). The product of the RSc1501 gene
(NP_519622) has I294, a bulky but not aromatic resi-
due, at the equivalent position followed by P295, a
rigid residue. It shows very low but measurable mono-
phenolase activity. The product of the RSc0337 gene
(NP_518458) has in that place a small residue, A241,
in agreement with the high tyrosine hydroxylase activ-
ity shown by this enzyme (Fig. 9).
This steric hindrance is one of the bases of the differ-
ence between monophenolases and o-diphenolases, but
Table 4. Monophenolase ⁄ o-diphenolase ratios of PPOs for carbox-
ylated ⁄ decarboxylated substrate pairs. DaO, Dopamine oxidase;
TaH, tyramine hydroxylase.
Enzyme
Optimum
pH TH ⁄ DO TaH ⁄ DaO

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

end of the CuB-binding site, tyrosinase-like enzymes
show an important variation. Usually there is another
H residue, but in some cases an L residue is found
(for instance, in the NP_519622 protein from R. solana-
cearum and in all animal tyrosinase-related proteins,
Tyrp1 and Tyrp2 [6,31]). Accordingly, the amino-acid
pair at that position is HH or LH. The variant residue
determines the putative interaction with the carboxylic
group on the side chain of the phenolic substrate, and
the affinity for the substrate. All enzymes with the HH
pair show high affinity for carboxylated substrates, but
all enzymes with LH show higher affinity for decarbox-
ylated substrates, as formerly reported [31] and con-
firmed in this work.
According to our proposal that the seventh histidine
is not the proton acceptor residue necessary to with-
draw the phenolic hydrogen for its co-ordination to
the CuB [32], another residue must obey this role. Its
identity is not known. Another proposed suggestion
for that role is an E residue based on its proximity to
the copper ions. This is E236 in sweet potato catechol
oxidase [26], equivalent to E
B1-4
in the general termin-
ology. However, according to Table 1, this residue is
conserved only in plant catechol oxidases. Tyrosinases
from Streptomyces have G at that position, animal
tyrosinases generally have Q, and mushroom tyrosin-
ase has I. These data indicate that many efficient tyro-
sinases carry out the catalysis without this acidic

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

His 279
His 307
Leu 306
Ile 294
His 102
9A: NP_518458 (gene RSc 0337)
9B: NP_519622 (gene RSc1501)
His 227
His 231
His 253
His 252
CuA CuB
CuA CuB
Fig. 9. A plausible scheme for the conformation of the active site
for the two PPOs presented here. The basic structure with the six
copper-binding H residues is drawn as described by Gerdemann
et al.[4] for catechol oxidase. Two main differences in R. solanacea-
rum PPOs are indicated. First, the residue preceding H
3B
, which is
H in NP_518458 and L in NP_519622. Second, the residue equival-
ent to the F261 of sweet potato catechol oxidase, responsible
for accessibility to CuA, which is probably occupied by A in
NP_518458 and I in NP_519622.
D. Herna
´
ndez-Romero et al. A novel tyrosinase from Rastonia solanacearum
FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 267
physiological role in the pathogenic process, it may
also be a novel biocatalyst useful in biotechnological

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

2
MoO
4
,5lm MnCl
2
, 0.5%
glycerol and 0.01% yeast extract in 50 mm sodium
phosphate buffer, pH 7. E. coli was routinely grown in
Luria–Bertani medium. When required, media were sup-
plemented with 50 lgÆmL
)1
kanamycin, ampicillin or rif-
ampicin, as necessary depending on the plasmid used and
selection needed.
To obtain routine bacterial extracts, cells were grown in
basal saline medium for 48 h to an absorbance of  1.2
and centrifuged at 5000 g for 10 min. The pellet was
washed with 0.9% NaCl solution, resuspended in 1 mL
0.1 m sodium phosphate, pH 7.0, containing 0.1 mm phe-
nylmethanesulfonyl fluoride plus a 1 : 500 dilution of ‘Pro-
tease inhibitor cocktail’Ò and disrupted by discontinuous
sonication by using a Braun Labsonic sonicator for
 4 min. The homogenate was centrifuged at 12 000 g for
4 min, and the supernatant was used for purification
and ⁄ or enzymatic activity determinations. Reagents for cell
culture media and enzymatic determinations were obtained
from Sigma Co. (St Louis, MO, USA).
Protein determination
Protein concentration was determined with the bicinchoninic
acid assay. Alternatively, A

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 ⁄

dopaminochrome per minute at 37 °C.
Enzyme purification
The extracts were concentrated using 15-mL Amicon ultra
Millipore centrifuge tubes of (cut-off 10 kDa). The prepara-
tions were loaded on a CM-Sephadex (Amersham Phar-
macia Biotech, Amersham, Bucks., UK) column (3 cm
diameter · 18 cm long). The column was eluted with
0.05 m sodium phosphate buffer, pH 7, until a volume
approximately equal to the total volume of the column had
been used. Once nonbound proteins were eluted from the
column, other proteins were eluted with a linear gradient of
NaCl up to 1.5 m in the same buffer. Fractions were
assayed for PPO activity.
SDS/PAGE
Enzyme purity was confirmed by SDS ⁄ PAGE [24]. Analyt-
ical dissociating SDS ⁄ PAGE was performed using 9%
acrylamide for the separating gel and 3% for the stacking
gel. The resolving buffer was Tris ⁄ HCl (pH 8.8), and the
reservoir buffer was Tris ⁄ glycine (pH 8.3), both containing
0.1% SDS. Samples were mixed in a 2 : 1 (v ⁄ v) ratio with
sample buffer (0.18 m Tris ⁄ HCl, pH 6.8, 15% glycerol,
0.075% bromophenol blue, 7.5% 2-mercaptoethanol and
9% SDS) and heated at 95 °C for 5 min before application.
Electrophoresis was run at 20 °C and a constant current of
15 mA for 20 min and 30 mA for  90 min. Protein bands
were visualized by Coomassie Brilliant Blue staining.
Reagents for SDS ⁄ PAGE were obtained from Bio-Rad
Laboratories (Richmond, CA, USA).
Acknowledgements
This work was supported in part by the project

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
References
1 Mason HS (1956) Structures and functions of the pheno-
lase complex. Nature 177, 79–81.
2 Van Gelder CW, Flurkey WH & Wichers HJ (1997)
Sequence and structural features of plant and fungal
tyrosinases. Phytochemistry 45, 1309–1323.
3 Solomon EI, Sundaran UM & Machonkin TE (1996)
Multicopper oxidases and oxygenases. Chem Rev 96,
2563–2605.
4 Gerdemann C, Eicken C & Krebs B (2002) The crystal
structure of catechol oxidase. New insights into the
functions of type-3 copper proteins. Acc Chem Res 35,
183–191.
5 Robb DA (1984) Tyrosinase. In Copper Proteins and
Copper Enzymes (Lontie R, ed.), Vol. 2, pp. 207–240.
CRC Press, Boca Raton, FL.
6 Garcı
´
a-Borro
´
n JC & Solano F (2002) Molecular anat-
omy of tyrosinase and its related proteins: beyond the

histidine-bound metal catalytic center. Pigment Cell Res
15, 162–173.
7 Thurston CF (1994) The structure and function of fun-
gal laccases. Microbiology 140, 19–26.
8 Lerch K & Ettlinger L (1972) Purification and charac-
terization of a tyrosinase from Streptomyces glaucescens.
Eur J Biochem 31, 427–437.
9 Huber M, Hintermann G & Lerch K (1985) Primary
structure of tyrosinase from Streptomyces glaucescens.
Biochemistry 24, 6038–6044.
10 Mercado-Blanco J, Garcı
´
a F, Ferna
´
ndez-Lo
´
pez M &
Olivares J (1993) Melanin production by Rhizobium
meliloti GR4 is linked to nonsymbiotic plasmid
pRmeGR4b: cloning, sequencing, and expression of the
tyrosinase gene. Mepa J Bacteriol 175, 5403–5410.
11 Lopez-Serrano D, Sanchez-Amat A & Solano F (2002)
Cloning and molecular characterization of a SDS-
activated tyrosinase from Marinomonas mediterranea.
Pigment Cell Res 15, 104–111.
12 Solano F, Lucas-Elı
´
o P, Ferna
´
ndez E & Sanchez-Amat

A (2000) Marinomonas mediterranea MMB-1 transposon
mutagenesis: isolation of a multipotent polyphenol oxi-
dase mutant. J Bacteriol 182, 3754–3760.
13 Sanchez-Amat A & Solano F (1997) A pluripotent poly-
phenol oxidase from the melanogenic marine Alteromo-
nas sp shares catalytic capabilities of tyrosinases and
laccases. Biochem Biophys Res Commun 240, 787–792.
14 Nosanchuk JD & Casadevall A (2003) The contribution
of melanin to microbial pathogenesis. Cell Microbiol 5,
203–223.
15 Kwon-Chung KJ & Rhodes JC (1986) Encapsulation
and melanin formation as indicators of virulence in
Cryptococcus neoformans. Infect Immun 51, 218–223.
16 Mayer AM & Harel E (1979) Polyphenol oxidases in
plants. Phytochemistry 18, 193–215.
17 Herna
´
ndez-Romero D, Solano F & Sanchez-Amat A
(2005) Polyphenol oxidase activities expression in Ral-
stonia solanacearum. Appl Environ Microbiol 11, 6808–
6815.
18 Kelley SK, Coyne VE, Sledjeski DD, Fuqua WC &
Weiner RM (1990) Identification of a tyrosinase from a
periphytic marine bacterium. FEMS Microbiol Lett 67,
275–279.
19 Salanoubat M, Genin S, Artiguenave F, Gouzy J,
Mangenot S, Arlat M, Billault A, Brottier P, Camus
JC, Cattolico L, et al. (2002) Genome sequence of the
plant pathogen Ralstonia solanacearum. Nature 415,
497–502.

20 Cooksey CJ, Garratt PJ, Land EJ, Pavel S, Ramsden
CA, Riley PA & Smit NPM (1997) Evidence of the
indirect formation of the catecholic intermediate sub-
strate responsible for the autoactivation kinetics of tyro-
sinase. J Biol Chem 272, 26226–26235.
21 Jara JR, Solano F & Lozano JA (1988) Assays for
mammalian tyrosinase: a comparative study. Pigment
Cell Res 1, 332–339.
22 Jime
´
nez-Cervantes C, Garcı
´
a-Borro
´
n JC, Valverde P,
Solano F & Lozano JA (1993) Tyrosinase isoenzymes in
mammalian melanocytes. Biochemical characterization
of two melanosomal tyrosinases from B16 mouse mela-
noma. Eur J Biochem 217 , 549–556.
23 Jime
´
nez-Cervantes C, Solano F, Kobayashi T, Urabe
K, Hearing VJ, Lozano JA & Garcı
´
a-Borro
´
n JC (1994)
A new enzymatic function in the melanogenic pathway:
the DHICA oxidase activity of tyrosinase related pro-
tein-1 (TRP1). J Biol Chem 269, 17993–18001.

24 Laemli UK (1970) Cleavage of structural proteins dur-
ing the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
25 Corpet F (1988) Multiple sequence alignment with hier-
archical clustering. Nucleic Acids Res 16, 10881–10890.
26 Klabunde T, Eicken C, Sacchettini JC & Krebs B
(1998) Crystal structure of a plant catechol oxidase con-
taining a dicopper center. Nat Struct Biol 5, 1084–1090.
27 Omura S, Ikeda H, Ishikawa J, Hanamoto A, Takaha-
shi C, Shinose M, Takahashi Y, Horikawa H,
Nakazawa H, Osonoe T, et al. (2001) Genome sequence
of an industrial microorganism Streptomyces avermitilis:
deducing the ability of producing secondary metabolites.
Proc Natl Acad Sci USA 98, 12215–12220.
28 Jime
´
nez-Cervantes C, Martı
´
nez-Esparza M, Solano F,
Lozano JA & Garcı
´
a-Borro
´
n JC (1998) Molecular
Interactions within the melanogenic complex. Forma-
tion of heterodimers of tyrosinase and TRP1 from
B16 mouse melanoma. Biochem Biophys Res Commun
253, 761–767.
29 Kobayashi T, Imokawa G, Bennett DC & Hearing VJ
(1998) Tyrosinase stabilization by Trp1 (the brown locus

protein). J Biol Chem 273, 31801–31805.
D. Herna
´
ndez-Romero et al. A novel tyrosinase from Rastonia solanacearum
FEBS Journal 273 (2006) 257–270 ª 2005 FEBS 269
30 Garcı
´
a-Molina F, Penalver MJ, Fenoll LG, Rodrı
´
guez-
Lopez JN, Varon R, Garcı
´
a-Ca
´
novas F & Tudela J
(2005) Kinetic study of monophenol and o-diphenol
binding to oxytyrosinase. J Mol Catal B Enzym 35,
185–192.
31 Olivares C, Garcı
´
a-Borro
´
n JC & Solano F (2002) Identi-
fication of active site residues involved in metal cofactor
binding and stereospecific substrate recognition in mam-
malian tyrosinase. Implications to the catalytic cycle.
Biochemistry 41, 679–686.
32 Tepper AW, Bubacco L & Canters GW (2005) Interac-
tion between the type-3 copper protein tyrosinase and
the substrate analogue p-nitrophenol studied by NMR.

J Am Chem Soc 127, 567–575.
33 Li H, Webb SP, Ivanivic J & Jensen JH (2004) Deter-
minants of the relative reduction potentials of type-1
copper sites in proteins. J Am Chem Soc 126,
8010–8019.
34 Kong KH, Hong MP, Choi SS, Kim YT & Cho SH
(2000) Purification and characterization of a highly
stable tyrosinase from Thermomicrobium roseum.
Biotechnol Appl Biochem 31, 113–118.
A novel tyrosinase from Rastonia solanacearum D. Herna
´
ndez-Romero et al.
270 FEBS Journal 273 (2006) 257–270 ª 2005 FEBS