Catechol Oxidase versus Tyrosinase Classification Revisited by Site-Directed Mutagenesis Studies

Article abstract of DOI:10.1002/anie.201902846

Catechol oxidases (COs) and tyrosinases (TYRs) are both polyphenol oxidases (PPOs) that catalyze the oxidation of ortho-diphenols to the corresponding quinones. By the official classification, only TYRs can also catalyze the hydroxylation of monophenols to ortho-diphenols. Researchers have been trying to find the molecular reason for the mono-/diphenolase specificity for decades. However, the hypotheses for the lack of monophenolase activity of plant COs are only based on crystal structures so far. To test these hypotheses, we performed site-directed mutagenesis studies and phylogenetic analyses with dandelion PPOs offering high phylogenetic diversity, the results of which refute the structure-based hypotheses. While plant PPOs of phylogenetic group 2 solely exhibit diphenolase activity, plant PPOs of phylogenetic group 1 unexpectedly also show monophenolase activity. This finding sheds new light upon the molecular basis for mono-/diphenol substrate specificity and challenges the current practice of generally naming plant PPOs as COs.

Full text of DOI:10.1002/anie.201902846

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Accepted Article
Title: Catechol Oxidase versus Tyrosinase Classification Revisited by
Site-Directed Mutagenesis Studies
Authors: Sarah Maria Prexler, Martin Frassek, Bruno Moerschbacher,
and Mareike Elisabeth Dirks-Hofmeister
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201902846
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Catechol Oxidase versus Tyrosinase Classification Revisited by
Site-Directed Mutagenesis Studies
Sarah M. Prexler[a], Martin Frassek[a], Bruno M. Moerschbacher*[a] and Mareike E. Dirks-Hofmeister[b]
Abstract: Catechol oxidases (COs) and tyrosinases (TYRs) are both
polyphenol oxidases (PPOs) that catalyze the oxidation of ortho-
diphenols to the corresponding quinones. By the official classification,
only TYRs can also catalyze the preceding hydroxylation of
monophenols to ortho-diphenols. As both enzymes cause undesired
browning of damaged fruits and vegetables and are of significant
interest for biotechnological applications, researchers have been
trying to find the molecular reason for the mono-/diphenolase
specificity for decades. However, the much-discussed hypotheses for
the lack of monophenolase activity of plant COs are almost
exclusively based on crystal structures so far, lacking experimental
evidence. To experimentally test these hypotheses, we used
dandelion PPOs offering high phylogenetic diversity to perform site-
directed mutagenesis studies. Our experimental and phylogenetic
analyses refute the crystal structure-based hypotheses. We found that
while plant PPOs of phylogenetic group 2 solely exhibit diphenolase
activity, plant PPOs of phylogenetic group 1 unexpectedly also show
monophenolase activity. This finding sheds new light upon the long-
discussed molecular basis for mono-/diphenol substrate specificity
and challenges the current practice of naming plant PPOs, as is the
rule, COs.
Scheme 1. Reactions catalyzed by catechol oxidases and tyrosinases.
was described in walnut (Juglans regia), ultimately invalidating
the hypothesis of these kingdom-specific enzymes[9]. Much effort
has since been made to elucidate the molecular differences
between enzymes showing both mono- and diphenolase activity
(i.e. TYRs) and enzymes showing only diphenolase activity
(i.e. COs).
The initial explanation for why COs lack monophenolase
activity was a bulky phenylalanine (F; HB3-13) atop of CuA
conserved in plant PPOs[10]. As deduced from the crystal structure
of sweet potato (Ipomoea batatas) PPO (IbCO; PDB ID: 1BT1),
this F may sterically hinder monophenols to turn for binding in the
catalytic cavity. An alternative hypothesis evolved from analyzing
the crystal structures of Bacillus megaterium TYR, where a
conserved water molecule was suggested to be activated by a
highly conserved glutamate (E; HB1-4) and an asparagine (N;
Catechol oxidases (COs; EC 1.10.3.1) and tyrosinases
(TYRs; EC 1.14.18.1) are ubiquitously distributed enzymes that
are e.g. responsible for the undesired browning of damaged fruits
and vegetables. Although they are of significant interest for
biotechnological applications, the molecular basis for their
different activities - either only on ortho-diphenols (COs) or
additionally on monophenols (TYRs) (Scheme 1) - is still unknown.
So far, COs and TYRs are considered as indistinguishable by
HB1+1)[11]
.
The activated water is thought to mediate
deprotonation of the monophenolic substrate necessary for its
hydroxylation. This observation was recently generalized to plant
PPOs, suggesting the E at position HB1-4 and the N at position
HB1+1 to be generally necessary and sufficient for
monophenolase activity[12]. This hypothesis was experimentally
approached by replacing G241 with N in VvPPOcs-3 (Vitis vinifera
‘Cabernet Sauvignon’) in an attempt to convert a CO into a TYR.
However, the qualitative, at best semi-quantitative in-gel activity
assay used showed that VvPPOcs-3-WT already possessed
some monophenolase activity[12,13]. Overall, both hypotheses for
the missing monophenolase activity of most plant PPOs require
further experimental evidence.
To biochemically assess the molecular basis for
mono-/diphenolase specificity, we performed site-directed
mutagenesis and detailed kinetic studies with well-characterized
dandelion (Taraxacum officinale) PPOs. Dandelion PPOs form
one of the largest PPO families ever discovered[14–21] consisting
of eleven isoenzymes separating into two different phylogenetic
groups (group 1 and group 2)[22]. Recently, it has been proposed
that plant PPOs in general divide into these two groups[23] whose
members differ in catalytic cavity architecture, kinetic parameters,
and substrate specificity. The latter has recently been shown to
be significantly impacted by the ‘substrate selector’ residue at
position HB2+1 in the active site[24]. We utilized the dandelion
PPOs’ phylogenetic diversity to experimentally investigate the
hypotheses for the lack of monophenolase activity of plant PPOs.
their amino acid sequences and physico-chemical properties[1,2]
.
Both enzyme classes belong to the super family of type-3 copper
proteins[3] characterized by the two copper binding motifs CuA
and CuB, each of them holding three histidine residues (HA1, HA2,
HA3 and HB1, HB2, HB3) able to coordinate one copper atom[4–6]
.
Together, the copper ions CuA and CuB can bind one oxygen
molecule.
COs were long believed to be solely plant enzymes, and all
plant PPOs were believed to be COs. Also, for a long time, TYRs
had only been found in fungi, animals and bacteria[7]. However, a
publication from 2010 was the first one to classify a fungal enzyme
from Aspergillus oryzae as a CO[8]. In 2014, the first plant TYR
[a]
[b]
Dr. S. M. Prexler, M. Frassek, Prof. Dr. B. M. Moerschbacher
Institut für Biologie und Biotechnologie der Pflanzen
Westfälische Wilhelms-Universität (WWU)
Schlossplatz 8, 48143 Münster (Germany)
E-mail: moersch@uni-muenster.de
Dr. M. E. Dirks-Hofmeister
WeissBioTech GmbH
An der Hansalinie 48-50, 59387 Ascheberg (Germany)
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. Locations of the mutated amino acid residues in the active site of PPO-2 (A-C) and PPO-6 (D-F), respectively. The PPO active site incorporates the
copper atoms A and B, each coordinated by three histidine residues (bright blue). In the illustrated met-state, the copper atoms together bind one hydroxide ion (red
sticks). A: PPO-2-WT with the wildtype amino acids that have been changed at position HB1+1 (G240) and at the ‘gate residue’ position HB3-13 (F260) in dark turquoise;
B: ‘gate residue’ mutant PPO-2-F260L (for PPO-2-F260P and PPO-2-F260G see Figure S1 in Supporting Information); C: HB1+1 mutant PPO-2-G240T (for PPO-2-G240
N
see Figure S1 in Supporting Information); D: PPO 6-WT with the wildtype amino acid that has been changed at position HB1+1 (T250) in dark turquoise; E: HB1+1
mutant PPO-6-T250N; F: HB1+1 mutant PPO-6-T250G. Brown: leucine (L); green: threonine (T); cyan: asparagine (N); yellow: glycine (G).
For this purpose, we chose one well-characterized enzyme from
each group (PPO-2 from group 1 and PPO-6 from group 2) for
heterologous expression in E. coli[25], site-directed mutagenesis
studies (Figure 1 and Figure S1 in Supporting Information), and
detailed kinetic characterization. The latter was performed with
the two mono-/diphenolic substrate pairs p-CO/4-MC and TA/DA
(Scheme 2), where the mono- and the diphenol were tested
separately.
Interestingly, PPO-2-WT showed clear activity with both
monophenolic substrates, whereas PPO-6-WT showed no
monophenolase activity at all (Figure 2, Table S2 in Supporting
Information). PPO-2-WT showed higher diphenolase than
monophenolase activity; the same was observed for Agaricus
bisporus TYR (AbTYR), the positive control enzyme for
monophenolase activity (Figure S2 and Table S2 in Supporting
Information). As PPO-2-WT possesses F at the ‘gate’ position
HB3-13 and no N at position HB1+1, this result already questions
both existing crystal structure-based hypotheses for the lack of
monophenolase activity. Still, we wanted to further test the
hypothesis that an N at position HB1+1 in combination with the
Scheme 2. Structures of the tested mono-/diphenol substrate pairs.
When we replaced E235 of PPO-2 with the sterically conservative,
but uncharged glutamine (Q), the enzyme lost all its activity
(Figure S3 in Supporting Information). When we replaced the
same residue with the similarly (i.e. negatively) charged
aspartate (D), activity was maintained (Figure S3 in Supporting
Information). This outcome confirms - in accordance with a study
by Hu et al.[26] - that a negatively charged residue is needed at
position HB1-4 in the active site of PPOs to act as catalytic base.
conserved E at position HB1-4 leads to monophenolase activity[12]
.
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Figure 2. Kinetic parameters of WT and mutein enzymes with the tested mono-/diphenol substrate pairs. All values (except for 2-WT and 2-FL) represent means
± standard deviations of the means calculated for three independent enzyme batches (see also Table S2 in Supporting Information). With each enzyme batch,
kinetics were performed three times, each in triplicates. The values of 2-WT and 2-FL represent the means ± standard deviations of three triplicate measurements
with one enzyme batch, as example of the performed measurements with two independent enzyme batches. For statistical evaluation, all values underwent a t-test
in SigmaPlot 12.5. Monophenols: p-cresol (p-CO) and tyramine (TA); diphenols: 4-methylcatechol (4-MC) and dopamine (DA); nd: not detected. *: p < 0.001.
Furthermore, we i) replaced the amino acid at position HB1+1 in
PPO-2 (G240) and in PPO-6 (T250) with N (leading to the muteins
PPO-2-G240N and PPO-6-T250N) and ii) exchanged the amino
acids at position HB1+1 of PPO-2 and PPO-6, leading to the
the ‘gate residue’ position: a leucine (L) found in wheat and
pineapple PPO as well as in filamentous fungus TYRs[19], a
proline (P) found in mushroom TYRs[19], and a glycine (G) found
in bacterial TYRs[27]. Again, all enzymes were successfully
expressed in E. coli in an active form and purified via Strep-tag
affinity chromatography (Figure S4 in Supporting Information).
Whereas the introduction of P260 and G260 led to total activity loss,
the F260L mutation reduced the total activity by two-thirds
(Figure 2, Table S2 in Supporting Information). Interestingly,
compared to PPO-2-WT, PPO-2-F260L showed the same KM
values and relative activities with all tested mono- and diphenolic
substrates except with DA (Figure 2, Table S2 and Figure S5 in
Supporting Information); with DA, PPO-2-F260L did not have any
activity. Hence, our results biochemically prove that the much-
discussed ‘gate residue’ F in plant PPOs does not affect
monophenolase activity at all. By contrast, the F at position HB3-13
muteins PPO-2-G240
T and PPO-6-T250G. Additionally, we
replaced the arginine (R) at position HB2+1 of PPO-6-T250G with
isoleucine (I) leading to the mutein PPO-6-T250G/R254I. The
reason for the construction of this double mutant was the strong
impact of R254 on the substrate specificity of PPO-6 recently
revealed[24]
.
All WT and mutein enzymes were successfully expressed in
E. coli in an active form and purified via Strep-tag affinity
chromatography (Figure S4 in Supporting Information). As
already observed for the WT enzymes, all PPO-2-based muteins
showed activity with both monophenolic substrates, whereas all
PPO-6-based muteins indicated no monophenolase activity at all
(Figure 2, Table S2 in Supporting Information). Still, the amino
acid residue at position HB1+1 seems to impact the enzymes’ total
activity and their ability to oxidize monophenols: Whereas G250
introduced into PPO-6 did not have any harmful effect on
diphenolase activity, T240 and N240 introduced into PPO-2 as well
as N250 introduced into PPO-6 reduced the total activity by two-
thirds. The total activity of PPO-6-T250G/R254I was even further
minimized. Regarding monophenolase activity, PPO-2-G240
T
showed strongly reduced activity, both with p-CO and TA
(Figure 2, Table S2 in Supporting Information). The introduction
of N240 did not increase PPO-2 monophenolase activity, and the
introduction of N250 into PPO-6 did not lead to its conversion from
a CO to a TYR.
To also approach the hypothesis about the bulky F
preventing monophenolase activity in plant PPOs, we replaced
F260 of PPO-2 with three amino acids occurring in other PPOs at
Figure 3. Monophenolase activity screening of E. coli strains expressing
dandelion PPOs. Mock: vector control.
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their phylogenetic grouping, fits previous reports on group-
specific differences in substrate specificity[24]
.
To view the correlation between mono-/diphenolase activity
and phylogenetic grouping more generally, we aligned and
phylogenetically analyzed the amino acid sequences of the TYR
domains of all plant PPOs that have, to date, been clearly
characterized for their natural mono-/diphenolase specificity
(Figure 4). After the split-up of moss PPOs and PPOs of higher
plants, the latter divide into two different phylogenetic groups: All
enzymes that exhibit both monophenolase and diphenolase
activity (except for LtLH) group into group 1, whereas all enzymes
showing only diphenolase activity group into group 2. The TYR
(+)-larreatricin hydroxylase (LtLH) specialized in the synthesis of
precursors of 8–8’ linked lignans specific to the creosote bush
Larrea tridentata[29] integrates in between group 1 and group 2
enzymes (Figure 4). Interestingly, both PPOs from Vitis vinifera
that have been characterized regarding mono-/diphenolase
specificity[12,30]
, clearly belong to group 1 PPOs. Whereas
VvPPOg was described earlier to exhibit both mono- and
diphenolase activity, VvPPOcs-3 was described to exhibit only
diphenolase activity. However, as mentioned before,
monophenolase activity was apparent, though weak, in the in-gel
Figure 4. Phylogenetic tree of plant PPOs. For the analysis, we aligned the TYR
domains of all plant PPOs that have, to date, been clearly biochemically
characterized for their natural mono-/diphenolase specificity. The tree was
rooted to a putative TYR sequence from the cyanobacterium Acaryochloris
marina (GenBank: ABW32074.1) as well as to putative PPO sequences from
the moss Physcomitrella patens (NCBI: XP_024397287.1) and the spike moss
Selaginella moellendorffii (NCBI: XP_024519810.1). The reliability of internal
branches was assessed by bootstrapping (1,000 replicates) and is indicated by
colored circles, with big red circles indicating 100 % support and small blue
circles indicating least support. All accession numbers are listed in Table S1 in
Supporting Information.
activity assay used in the correspondent study[13]
.
In the amino acid alignment, we found clear differences
between group 1 and group 2 enzymes, namely in the residues
flanking the CuB coordinating histidines (HB1+1 and HB2+1) and in
the residues HA1+5, HA1+9 and HB3-8 (Figure S7 in Supporting
Information). The residues located at HB1+1 and HB2+1 have
previously been proposed to impact substrate specificity in plant
PPOs[13,24]. Another distinctive feature in the amino acid alignment
is an inserted stretch of four to seven (IbCO: one) amino acids in
front of the CuB motif of group 2 enzymes, as already described
by Molitor et al.[23]. As the inserted residues form a surface-
exposed loop structure near the active site, they are likely to
influence enzyme-substrate interactions.
In summary, by site-directed mutagenesis and detailed kinetic
characterization, we biochemically disproved the current
hypotheses that suggest that plant PPOs lack monophenolase
activity due to a missing N at position HB1+1 and the hindering
‘gate residue’ F. Using the dandelion PPOs’ phylogenetic diversity,
we have shown that plant PPOs exhibiting monophenolase
activity normally belong to phylogenetic group 1, whereas those
exhibiting solely diphenolase activity generally belong to group 2.
This finding adds new information that can be used in future
studies to better assess the molecular basis for mono-/diphenol
substrate specificity of PPOs; simultaneously, it challenges the
current practice of naming plant type-3 copper enzymes, as is the
rule, COs.
of plant PPOs seems to be necessary for total enzyme activity. In
our protein modeling studies, we observed a ‘sandwich structure’
of the second CuB coordinating histidine HB2, the catechol ring of
the substrate and the ‘gate residue’ F. Hence, we propose that
hydrophobic interactions between the phenolic substrate’s π
electron system and the π electron systems of HB2 and F lead to
correct positioning and stabilization of the substrate in the
catalytic cavity (see also [24]). This deduction is in accordance with
a hypothesis by Bijelic et al., who attributed the F at position
HB3-13 of walnut TYR to a ‘substrate-guiding’ function[28]
.
Our biochemical studies disprove the crystal structure-
based hypotheses that have tried to explain why plant PPOs lack
monophenolase activity. Our data in fact point out that
phylogenetic group 1 plant PPOs seem to have monophenolase
activity that is unaccounted for in their original classification,
whereas group 2 plant PPOs seem not to have monophenolase
activity. To check this hypothesis within the dandelion PPO family,
we screened all PPOs available in expression strains for
monophenolase activity. Indeed, PPO-1, PPO-2 and PPO-3
(group 1) showed clear activity with TA, whereas PPO-4, PPO-6,
PPO-7 and PPO-9 (group 2) did not (Figure 3). Additionally, we
quantitatively measured the specific activity of a second pair of
purified group 1 and group 2 enzymes, PPO-1 and PPO-7, with
the two mono-/diphenolic substrate pairs p-CO/4-MC and TA/DA:
Whereas PPO-1 showed monophenolase activity comparable to
the one of PPO-2, no monophenolase activity was detected for
PPO-7 (Figure S6 in Supporting Information). This indication, that
the mono-/diphenolase activity of dandelion PPOs is correlated to
Acknowledgements
Sarah M. Prexler greatly appreciates sponsoring by the
Scholarship Program of the German Federal Environmental
Foundation (Deutsche Bundesstiftung Umwelt, Osnabrück,
Germany). The authors thank Kai H. Edel for his support
regarding phylogenetic analysis and Celeste R. Brennecka from
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the Science Writing Support Service of WWU for her editorial
support.
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Sarah M. Prexler, Martin Frassek, Bruno
M. Moerschbacher*, Mareike E. Dirks-
Hofmeister
Catechol Oxidase versus Tyrosinase
Classification Revisited by Site-
Directed Mutagenesis Studies
The much-discussed hypotheses about the residue at position HB1+1 and the ‘gate residue’ phenylalanine being potentially responsible
for the assumed general lack of monophenolase activity of plant polyphenol oxidases (PPOs) are tested by site-directed mutagenesis
studies with dandelion (Taraxacum officinale) PPOs. The experimental results as well as additional phylogenetic analyses refute both
hypotheses. Instead, we found that plant PPOs of phylogenetic group 2 solely exhibit diphenolase activity and, hence, are catechol
oxidases (COs) as expected, while plant PPOs of phylogenetic group 1 additionally show monophenolase activity and, therefore, are
actually tyrosinases (TYRs).
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