Significant improvement of oxidase activity through the genetic incorporation of a redox-active unnatural amino acid

Article abstract of DOI:10.1039/c5sc01126d

While nature employs various covalent and non-covalent strategies to modulate tyrosine (Y) redox potential and pK_a in order to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Through the genetic incorporation of 3-methoxytyrosine (OMeY) into myoglobin, we replicated important features of cytochrome c oxidase (CcO) in this small soluble protein, which exhibits selective O₂ reduction activity while generating a small amount of reactive oxygen species (ROS). These results demonstrate that the electron donating ability of a tyrosine residue in the active site is important for CcO function. Moreover, we elucidated the structural basis for the genetic incorporation of OMeY into proteins by solving the X-ray structure of OMeY specific aminoacyl-tRNA synthetase complexed with OMeY.

Full text of DOI:10.1039/c5sc01126d

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Significant Improvement of Oxidase Activity through
the Genetic Incorporation of a Redox-active
Unnatural Amino Acid
Cite this: DOI: 10.1039/x0xx00000x
c‡
b‡
a,b‡
b
b
b
Yu Yang , Qing Zhou , Li Wang , Xiaohong Liu , Wei Zhang , Meirong Hu ,
Received 00th January 2012,
Accepted 00th January 2012
b
a,b
b
c
b
b
Jianshu Dong , Jiasong Li , Lv Xiaoxuan , Hanlin Ouyang , Han Li , Feng Gao ,
Weimin Gong , Yi Lu* , Jiangyun Wang*
b
c
b
DOI: 10.1039/x0xx00000x
While nature employs various covalent and non-covalent strategies to modulate tyrosine (Y)
redox potential and pK to optimize enzyme activities, such approaches have not been
www.rsc.org/
a
systematically applied for the design of functional metalloproteins. Through the genetic
incorporation of 3-methoxytyrosine (OMeY) into myoglobin, we recapitulated important
features of cytochrome c oxidase (CcO) into this small soluble protein, which exhibits
selective O
2
reduction activity while generating small amount of reactive oxygen species
(
ROS). These results demonstrate that the electron donating ability of a tyrosine residue in the
active site is important for CcO function. Moreover, we elucidated the structural basis for the
genetic incorporation of OMeY into proteins, by solving the X-ray structure of OMeY specific
aminoacyl-tRNA synthetase in complex with OMeY.
enzyme activity.10-16 However, it remains difficult to perform
rational tuning of redox potential and pK of specific tyrosine
a
Introduction
Designing artificial enzymes with higher activity and selectivity residues in designed metalloproteins. Here we show, through the
can reveal important features responsible for tuning enzymatic genetic incorporation of redox-active unnatural amino acids with
activities, and result in efficient catalysts for practical desirable redox potential, significant improvement of oxidase
applications.1-9 One key mechanism which accounts for the high activity can be achieved.
activity of many natural enzymes is the fine-tuning of the redox
During the final stage of aerobic respiration, CcO catalyzes the
potential of tyrosine residues. In order to optimize electron efficient reduction of O to H O, which requires rapid transfer of
2
2
transfer rate to enable enzymatic turnover with high efficiency four electrons and four protons to the oxygen substrate,
and selectivity, nature has exploited various strategies, such as preventing the release of toxic reactive oxygen species (ROS).15
post-translational modifications including topa quinone in The key step in oxygen reduction is the scission of the O-O bond
copper amine oxidases, Tyr-His crosslink in CcO, Tyr-Cys in the ferric–superoxo intermediate, leading to the formation of
1
7
crosslink in galactose oxidases (GO), and histidine base an intermediate P in the heme a /Cu binuclear active site. The
3
B
association in photosystem II (PSII).10-16 Such strategies are donor of a proton and electron for this reaction has been
highly effective in modulating the redox potential (from 0.1 V to suggested to be a unique tyrosine residue covalently cross-linked
a
B
1.1 V vs. NHE) and pK of specific tyrosine residues to suit for to one of the histidine ligands of Cu . This Tyr-His crosslink is
specific needs of various reactions, therefore greatly enhancing thought to lower the pK and redox potential of the tyrosine
a
residue, thus facilitating proton and electron donation to the
a
School of Life Sciences, University of Science and Technology of
oxygen substrate.15, 18
China, Hefei, Anhui, 230026, China.
b
In previous studies, we have reported the introduction of
various tyrosine analogs into a myoglobin-based functional
oxidase,19, 20 including imiTyr,19 which mimics the Tyr-His
cross-link in CcO, and a series of halogenated Tyr with
decreasing pK
a
.21 By replacing Tyr33 with imiTyr and
Laboratory of RNA Biology, Institute of Biophysics, Chinese
Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing,
1
00101, China. Email: jwang@ibp.ac.cn
Center of Biophysics and Computational Biology and Department
c
of Chemistry, University of Illinois at Urbana-Champaign, Urbana,
Illinois, 61801, USA. Email: yi-lu@illinois.edu
‡
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available:
halogenated tyrosine analogs, activity and selectivity of the
functional oxidase increases. Moreover, the oxidase activity is
†
Experimental procedures. See DOI: 10.1039/b000000x/
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a
correlated with the pK of the phenol ring of Tyr or its S4). DNA sequencing revealed that this clone contains the
halogenated analogs, suggesting the active role of Tyr in oxidase Met379Val mutation. Molecular modeling indicates that the
reaction. However, since reduction potentials of halogenated Tyr Met379Val mutation results in significant enlargement of the
a
are closely related to their pK , we have not yet addressed enzyme pocket to allow for optimal interaction between the
whether fine-tuning of the redox potential of the tyrosine residue enzyme and the OMeY substrate (Fig. S5). The reduction
influences oxidase activity. Herein we report genetic potentials of OMeY is 179mV lower than that of Tyr at pH 7,
incorporation of a tyrosine analog, 3-methoxy tyrosine (OMeY), whereas the pKa of OMeY and tyrosine are similar (Fig. S2 and
a
that has lower reduction potential but similar pK compared to S6).
Tyr, to provide evidence that reduction potential tuning of Tyr is
also important for oxidase activity.
To incorporate OMeY at defined sites selectively in proteins, a
mutant Methanocaldococcus jannaschii tyrosyl amber
suppressor tRNA (MjtRNATyrCUA)/tyrosyl-tRNA synthetase
(MjTyrRS) pair was evolved that uniquely specifies OMeY in
response to the TAG codon, as previously reported.19 The
evolved TyrRS (Fig. S7-S8), named OMeYRS, has six
mutations: Tyr32Glu, Leu65Ser, His70Gly, Tyr109Gly,
Asp158Asn, and Leu162Val.
Results and discussion
We first attempted to genetically incorporate two previously
reported Tyr analogs, 3,4-dihydroxy-l-phenylalanine (Dopa)22
and 3-amino-tyrosine (NH
2
Y),23 which have lower redox
potentials than tyrosine, into the 33rd position of Cu
B
Mb.
To determine if OMeY is incorporated into protein with high
efficiency and fidelity, an amber stop codon was substituted for
Ser4 in sperm whale myoglobin (Mb). Protein production was
carried out in E. coli in the presence of the selected synthetase
However, yields of these mutant proteins are quite low,
preventing further characterization. One explanation for these
results is that Dopa and NH Y can both undergo irreversible two-
2
electron oxidation reaction to afford dopaquinone, which is well-
known to be highly reactive and play pivotal roles in
melanogenesis (Fig. S1).24 This problem may exacerbate when
an oxidase or oxidase-mimicking enzyme is over-expressed in E.
coli. In order to circumvent this problem, we decided to
genetically incorporated OMeY, because its redox potential at
pH 7 is 179 mV lower than that of Tyr (Fig. S2), but can’t easily
undergo two-electron oxidation reaction.
(
OMeYRS), MjtRNATyrCUA and 1 mM OMeY, or in the absence
of OMeY as a negative control. Analysis of the purified protein
by SDS-PAGE showed that full-length myoglobin was
expressed only in the presence of OMeY (Fig. 1A), indicating
that OMeYRS was specifically active with OMeY but inactive
with natural amino acids. The yield for this mutant myoglobin
was 10 mg/L. For comparison, the yield of wild-type sperm
whale myoglobin (WTMb) was 50 mg/L. ESI-MS analysis of the
Ser4 OMeY mutant myoglobin gave an observed average mass
of 18461.6 Da, in agreement with the calculated mass of
18461.1Da (Fig. 1B).
Scheme 1. A biosynthetic route to OMeY, catalyzed by the TPL
mutant M379V.
The difficulty in synthesizing unnatural amino acid analogs of
Tyr, such as OMeY, is often a limiting factor for systematic
investigation of the role of Tyr in different enzymes. Using
enzymatic transformation by native or engineered tyrosine
phenol lyase (TPL), as demonstrated herein, has been proven to
be a powerful tool to synthesize tyrosine analogs.25, 26 To
synthesize OMeY, we first tried to transform 2-methoxyphenol
to OMeY by using the wild type Symbiobacterium sp. SC-1 TPL,
which is more thermostable than Citrobacter freundii
(
ATCC8090).27 However, we could not detect any OMeY by
ninhydrin thin-layer chromatography (TLC) assay. To evolve a
TPL mutant that can efficiently catalyze this tranformation, we
screened a TPL library pEt-SymbTPL, which harbors random
mutations at sites Phe36, Met288, Met379, and Phe448 as
previously reported,26, 28 and found that one clone efficiently
catalyzed the synthesis of OMeY, as confirmed by mass
spectrometry, after purification of the product by HPLC (Fig. S3-
Fig. 1 A) SDS-PAGE of expression of TAG4 myoglobin mutant
in the presence(right lane) and absence(middle lane) of 1mM
unnatural amino acid OMeY (UAA). B) ESI-MS of the TAG4
2
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mutant. The inset shows the deconvoluted spectrum; expected Cu
mass: 18461 Da, found: 18461.62 Da.
B
Mb was able to catalyze O
turnovers without significant reduction of catalytic rate. Under
2
reduction for more than 1100
similar conditions, Phe33Tyr-Cu Mb could only catalyze the
B
To test whether the catalytic activity could be improved through reaction for less than 500 turnovers.20
the genetic incorporation of unnatural amino acids, we replaced
Phe33 in Cu
B B
Mb with OMeY, generating Phe33OMeY-Cu Mb
(
Fig. 2). This mutant showed similar UV-Vis spectrum to that of
B
the Phe33Tyr-Cu Mb (Fig. S9), indicating that the overall
environment around the heme center should also be similar.20
Fig. 2 Structural model of OMeY myoglobin mutant, constructed
based on the crystal structure of the Phe33Tyr-Cu
FWX).
B
Mb (pdb code
4
We then measured the rates of oxygen reduction catalyzed by 6
μM myoglobin mutants with an O electrode in 20 mM
tris(hydroxymethyl)aminomethane (Tris) buffer at pH 7.4.
Ascorbate (1000 equivalents) and tetramethyl-p-
2
phenylenediamine dihydrochloride (TMPD, 100 equivalents)
were used as reductant and redox mediator respectively.29 To
differentiate between reactive oxygen species (ROS) and water
products, we used catalase and superoxide dismutase (SOD),
which catalyzed the disproportionation of hydrogen peroxide or
superoxide into oxygen and water. If O
the formation of ROS but not water, the O
decrease in the presence of catalase and SOD, because they will
convert ROS to O . By comparing the rates of reduction in the
presence and absence of ROS scavenger, the portion of O
2
consumption results in
2
reduction rate should
2
2
reduction due to water formation (in blue) and due to ROS
formation (in red) can be calculated (Fig. 3A and Table S2). Our
results show that Phe33Tyr-Cu
rate of 6.5 μM/min, with 51% of O
In contrast, Phe33OMeY-Cu Mb exhibited significant higher
oxidase activity at 15.0 μM/min for O reduction, 82% of which
was converted to water. Similar to the case of Phe33Tyr-Cu Mb,
addition of copper into Phe33OMeY-Cu Mb does not increase
oxidase activity.20 Since the pK
of OMeY (Fig. S6) is similar to
that of Tyr, the lower redox potential of OMeY should be
responsible for increased oxidase activity and O reduction
selectivity of Phe33OMeY-Cu Mb.
To further demonstrate the robustness of the best oxidase-
mimicking enzyme, Phe33OMeY-Cu Mb, we carried out
B
Mb was able to reduce O
2
at a
2
being converted into water.
B
2
B
Fig. 3 A) Rates of oxygen reduction to form either water (blue)
or ROS (red) catalyzed by 6μM of WTMb, Phe33Tyr-CuBMb or
B
a
Phe33OMeY-Cu
Phe33OMeYCu
. C) Plot of oxidase activity of Phe33Tyr-Cu
Cu Mb, Phe33F Y-Cu Mb, Phe33F Y-Cu
Phe33OMeY-Cu Mb vs. peak potential at pH 7 (E
corresponding Tyr and Tyr analogs. E
B
Mb. B) O
Mb measured during the stepwise addition of
Mb, Phe33ClY-
2
-reduction turnover number by
B
2
O
2
B
B
B
2
B
3
B
Mb,
and
) of the
B
p
B
p
measured by cyclic
multiple turnover experiment (Fig. 3B). The Phe33OMeY-
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voltammetry is 941 mV for Tyr, 847 mV for ClY, 782 mV for
Tyr, 860 mV for F Y, and 762 mV for OMeY. Abbreviations: Notes and references
F
2
3
ClY, 3-chlorotyrosine; F
2 3
Y, 3,5-difluorotyrosine; F Y, 2,3,5-
difluorotyrosine.
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Acknowledgements
We gratefully acknowledge the Major State Basic Research
Program of China (2015CB856203, 2011CBA00800), National
Science Foundation of China (21325211, 90913022, 31270859
21473237) to J.W., and National Institutes of Health (GM06221)
to Y.L.
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