Kinetic and Structural Analysis of Two Linkers in the Tautomerase Superfamily: Analysis and Implications

Article abstract of DOI:10.1021/acs.biochem.1c00220

The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple β-α-β structural scaffold. Most members are constructed from a single-core β-α-β motif or two consecutively fused β-α-β motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.

Full text of DOI:10.1021/acs.biochem.1c00220

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Article
Kinetic and Structural Analysis of Two Linkers in the Tautomerase
Superfamily: Analysis and Implications
Bert-Jan Baas, Brenda P. Medellin, Jake A. LeVieux, Kaci Erwin, Emily B. Lancaster,
William H. Johnson, Jr., Tamer S. Kaoud, R. Yvette Moreno, Marieke de Ruijter, Patricia C. Babbitt,
Yan Jessie Zhang, and Christian P. Whitman*
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ABSTRACT: The tautomerase superfamily (TSF) is a collection
of enzymes and proteins that share a simple β−α−β structural
scaffold. Most members are constructed from a single-core β−α−β
motif or two consecutively fused β−α−β motifs in which the N-
terminal proline (Pro-1) plays a key and unusual role as a catalytic
residue. The cumulative evidence suggests that a gene fusion event
took place in the evolution of the TSF followed by duplication (of
the newly fused gene) to result in the diversification of activity that
is seen today. Analysis of the sequence similarity network (SSN)
for the TSF identified several linking proteins (“linkers”) whose
similarity links subgroups of these contemporary proteins that
might hold clues about structure−function relationship changes accompanying the emergence of new activities. A previously
uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase
(
4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of
active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT
activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other
characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced
hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11.
Crystallographic analysis provides a structural context for these observations.
6
7,8
7
inkers are proteins that have sequence similarity with
rization, hydrolytic dehalogenation, hydration, decarbox-
9
10
11
L
other proteins in different subgroups in a sequence
ylation, aldol condensation, and oxygenation. Members
also have biological activities where the best characterized one
is the macrophage migration inhibitory factor (MIF) that
1
similarity network (SSN). Although these linking sequences
are contemporary ones, they can provide hints about
intermediate or transitional features resulting in the divergence
from a common ancestor. Characterization of the proteins
coded by linking sequences reveals changes in structure and
function that might have occurred in the evolution of proteins
in one cluster from the proteins in another cluster. The
potential value of examining relationships among such
contemporary linkers was demonstrated in the analysis of the
12
functions as a pro-inflammatory cytokine.
A global analysis of the TSF collected more than 11000
nonredundant sequences of proteins found throughout the
biosphere. These sequences were sorted into five subgroups
4-oxalocrotonate tautomerase, 4-OT, 5-(carboxymethyl)-2-
hydroxymuconate isomerase, CHMI, malonate semialdehyde
decarboxylase, MSAD, cis-3-chloroacrylic acid dehalogenase,
cis-CaaD, and MIF] in the SSN that aligned with the
characterized activities of the title enzymes. Although the
SSN showed several linkers connecting the subgroups, four
1
[
1
SSN for the tautomerase superfamily (TSF).
1
Members of the tautomerase superfamily are constructed
from a β−α−β structural unit that is found as a single unit
(
1
58−84 amino acids) or two consecutively fused units (110−
1
−3
50 amino acids).
The experimentally characterized
Received: March 25, 2021
Revised: May 2, 2021
Published: May 21, 2021
members have a catalytic amino-terminal proline (Pro-1)
that functions as a general base or acid, depending on the pK
a
3
−5
of the prolyl nitrogen.
This simple motif has been
repurposed by nature many times over to catalyze a variety
of functionally diverse enzymatic reactions, including tautome-
©
2021 American Chemical Society
https://doi.org/10.1021/acs.biochem.1c00220
1
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linker enzymes along a similarity path between the 4-OT and
OT-like subgroup. Notably, tautomerase activity with the
dicarboxylate substrate was lost, but that with the mono-
carboxylate substrate was retained. The final series of hops
cis-CaaD subgroups were chosen for initial characterization as a
1
,13
proof of principal (Figure 1).
The structure-based
(
linker 1 to CgX, a cis-CaaD homologue that functions as an
inefficient cis-CaaD, and CgX to cis-CaaD designated CC in
1
Figure 1) complete the transition.
Besides the linker 1 and linker 2 representative nodes linking
the 4-OT and cis-CaaD subgroups, the SSN shows a second
edge that also connects these subgroups. The representative
nodes for this second edge are designated N1 and N2 (Figure
1
). Hence, N1 (in the cis-CaaD subgroup) and N2 (in the 4-
OT subgroup) were chosen for sequence, kinetic, mechanistic,
and structural analysis. N1 has the key components of the
catalytic machinery (Pro-1, His-28, Arg-70, Arg-73, Tyr-103,
3
,8
and Glu-114) found in cis-CaaD. Crystallographic analysis of
N1 reveals that the active site mirrors those of two other
characterized cis-CaaD subgroup members along with cis-
CaaD. Not surprisingly, kinetic analysis and NMR analysis
show that N1 functions similarly with pronounced hydration
activity, but no 4-OT activity. Like the others, tautomerase
activity is exhibited with a monocarboxylate substrate,
phenylenolpyruvate. N2 lacks Arg-39, a key residue for 4-OT
1
,3,14
activity, although Pro-1 and Arg-11 are present.
Kinetic
analysis shows that 4-OT activity is diminished in comparison
to those of other characterized members of the 4-OT
subgroup. Covalent modification of the Pro-1 nitrogen by 2-
oxo-3-pentynoate (2-OP) reflects a low pK value, which is also
a
3
−5
a property shared with other 4-OT subgroup members.
However, unlike other characterized members of the subgroup,
hydratase activity is enhanced in N2, which might be due to
the presence of Arg-8 along with Arg-11. Crystallographic
analysis of N2 provides a structural context. The weight of the
experimental evidence highlights the importance of Arg-8 in
Figure 1. Close-up of the 50% representative sequence similarity
network of the TSF showing representative nodes that connect the 4-
OT and cis-CaaD subgroups (modified from ref 1). The
representative nodes of the 4-OT subgroup are colored brown
15
the evolution of a 4-OT-like protein to cis-CaaD.
(
bottom), where 4-OT is designated by the yellow triangle, and those
in the cis-CaaD subgroup are colored cyan, where cis-CaaD (CC) is
designated by the yellow triangle (top). The other nodes that contain
biochemically characterized enzymes are labeled using the abbrevia-
tions below. Large unlabeled squares designate representative nodes
that contain structurally characterized protein(s). Two previously
uncharacterized linker nodes, N1 and N2, are the targets of this study.
The structural changes associated with a series of hops along the
similarity pathway between the 4-OT and cis-CaaD subgroups are
EXPERIMENTAL PROCEDURES
■
Materials. Chemicals, biochemicals, buffers, and solvents
were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO), Fisher Scientific, Inc. (Pittsburgh, PA), EMD Millipore,
Inc. (Billerica, MA), or Bio-Rad Laboratories, Inc. (Hercules,
CA), unless otherwise noted below. Phenylenolpyruvate (PP)
was purchased from Fluka Chemical Corp. (Milwaukee, WI) as
the crystalline free acid, which exists exclusively in the enol
form. Propiolic acid (98%) and 2-butynoate were purchased
from Sigma-Aldrich and purified further prior to use by
distillation and recrystallization, respectively. 2-Hydroxymuco-
1
summarized below and described in detail elsewhere. Abbreviations:
F4OT, fused 4-OT; L2, linker 2; L1, linker 1; CgX, a cis-CaaD
homologue.
6
14
comparison of these linkers identified transitions between
them that were paralleled in the kinetic analysis and suggested
how the cis-CaaD-like enzymes might evolve from a 4-OT-like
nate (2-HM), 2-oxo-3-pentynoate (2-OP), and 2,3-
16
butadienoate were synthesized according to the indicated
references. The diethylaminoethyl (DEAE) Sepharose fast flow
resin and the PD-10 Sephadex G-25 M buffer-exchange
column were obtained from GE Healthcare Bio-Sciences
(Pittsburgh, PA). The Econo-Column chromatography col-
umns and the Bio-Gel P60 gel beads used for size-exclusion
chromatography were obtained from Bio-Rad Laboratories,
Inc. The Spectra/Chrom LC column was obtained from
Spectrum Laboratories, Inc. (Rancho Dominguez, CA). The
Amicon stirred cell concentrators and the ultrafiltration
membranes (10000 Da, MW cutoff) were purchased from
EMD Millipore, Inc. InstantBlue for staining SDS−PAGE gels
was obtained from C.B.S. Scientific Company, Inc. (Del Mar,
CA).
1
,13
ancestor.
In a stepwise series of “hops” along the similarity pathway,
the first hop connected the short 4-OT to a fused 4-OT
(
F4OT), which represents the fusion of two β−α−β structural
1
units. The next hop connected the fused 4-OT to linker 2
also a fused 4-OT) designated L2 (in Figure 1). Both linker 2
(
and fused 4-OT showed structural and kinetic similarities with
other members of the 4-OT subgroup, including the
conservation of key active site residues and robust activities
using 2-hydroxymuconate (2-HM) and phenylenolpyruvate
1
,4−6
(
PP), di- and monocarboxylate substrates, respectively.
The hop from linker 2 to linker 1 (designed L1 in the cis-CaaD
subgroup) introduced active site features associated with cis-
CaaD-like properties and a loss of those associated with the 4-
Bacterial Strains, Plasmids, and Growth Conditions.
Escherichia coli strain BL21-Gold(DE3) was obtained from
1
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Table 1. Crystallographic Data Collection and Refinement Statistics
N1
N2
Data Collection
space group
P2 2 2
1 1
R32
1
cell dimensions
a, b, c (Å)
α, β, γ (deg)
resolution (Å)
wavelength (Å)
41.36, 87.82, 120.71
90.00, 90.00, 90.00
50.00−2.45 (2.49−2.45)
78.84, 78.84, 182.95
90.00, 90.00, 120.00
50.00−1.65 (1.68−1.65)
a
a
0.97648
0.127 (0.649) /0.064 (0.368)
0.886 (0.645)
12.2 (1.6)
90.4 (82.1)
4.5 (3.9)
0.97648
0.057 (0.616) /0.018 (0.290)
0.962 (0.767)
50.2 (1.9)
a
a
a
a
Rsym/R
CC1/2
pim
b
a
a
a
a
I/σ
a
a
completeness (%)
redundancy
99.6 (96.7)
a
a
10.6 (5.3)
Refinement
a
a
resolution (Å)
no. of unique reflections
Rwork
31.80−2.45 (2.53−2.45)
38.00−1.65 (1.71−1.65)
a
a
15278 (1336)
26626 (2544)
0.1752 (0.2436)
a
a
0.1954 (0.2439)
0.2633 (0.3320)
c
a
a
Rfree
0.1865 (0.2668)
no. of atoms
protein
3396
0
69
984
0
91
ligand/ion
water
2
B-factor (Å )
protein
ligand/ion
33.9
0
25.4
0
water
35.8
34.4
root-mean-square deviation
bond lengths (Å)
bond angles (deg)
Ramachandran plot (%)
favored
0.010
1.25
0.006
0.73
97.64
1.65
0.71
100
0
allowed
d
outliers
0
e
e
f
e
f
Molprobity score
1.7 /98th percentile
0.89 /100th percentile
a
b
Values for the corresponding parameters in the outermost shell in parentheses. CC is the Pearson correlation coefficient for a random half of
1
/2
c
the data; the two numbers represent the lowest- and highest-resolution shell, respectively. R is the Rwork calculated for ∼10% of the reflections
randomly selected and omitted from refinement. There are three Ramachandran outliers in N1, which correspond to aspartate residues (Asp-13
chain A, Asp-13 chain B, and Asp-13 chain C) all with strong electron density. The MolProbity score is calculated by combining the Clashscore
with rotamer and Ramachandran percentage and scaled on the basis of X-ray resolution. The percentage is calculated with the 100th percentile as
free
d
e
f
the best and the 0th percentile as the worst among structures of comparable resolution.
Agilent Technologies (Santa Clara, CA). The genes encoding
N2 (4-OT subgroup) from Gammaproteobacteria bacterium
SG8_31 (UniProt entry A0A0S8FF56) and N1 (cis-CaaD
subgroup) from Corynebacterium halotolerans YIM 70093
using the ion trap detector or nanoflow liquid chromatograph
and deconvoluted using Thermo Protein Deconvolution
7
software. Samples were prepared as described elsewhere.
Discovery of N2 and N1. The SSN for the TSF was
1
(
UniProt entry M1NLA4) were codon-optimized for ex-
constructed as described elsewhere. N2 and N1 are
pression in E. coli, synthesized, and cloned into expression
vector pJ411 by ATUM (Newark, CA). Cells were grown
overnight (∼16 h) at 37 °C in Luria-Bertani (LB) medium,
supplemented with 25 mM Na HPO and 25 mM KH PO ,
representative nodes for a second representative edge
connecting the 4-OT and cis-CaaD subgroups. N1 was
included in the phylogenetic tree of the 4-OT and cis-CaaD
linkers, and both were included in the linker control
2
4
2
4
1
buffered to pH ∼6.75, 5 mM Na SO , 2 mM MgSO , and
experiments described elsewhere.
2
4
4
kanamycin (Kn, 30 μg/mL).
Expression and Purification of N2 and N1. N2 and N1
were expressed and purified as follows. Fresh BL21Gold(DE3)
transformants containing expression vector pJ411-N2 or
pJ411-N1 were collected from a LB/Kn plate and used to
inoculate a preculture (LB/Kn medium, 30 μg/mL, 10 mL).
After being grown for ∼6 h at 37 °C, the preculture was used
to inoculate 1 L of the LB supplemented medium. After being
grown overnight at 37 °C while being shaken (250 rpm), the
culture was incubated at 22 °C for 1 h and subsequently made
0.5 mM in isopropyl β-D-thiogalactoside to induce expression.
After being incubated for 5 h at 22 °C, the cells were harvested
General Methods. Steady-state kinetic assays were
performed on an Agilent 8453 diode-array spectrophotometer
at 22 °C. Nonlinear regression data analysis was performed
using Grafit (Erithacus Software Ltd., Staines, U.K.). Protein
17
concentrations were determined by the Waddell method.
SDS−PAGE was carried out on denaturing gels containing
18
1
2% polyacrylamide. Mass spectrometry was conducted by
The University of Texas at Austin Proteomics Facility using an
Optimize Technologies protein trap in line with a Thermo
Orbitrap Fusion mass spectrometer. The data were collected
1
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by centrifugation (10 min, 2500g) and resuspended in 10 mM
Na HPO buffer (pH 7.3, buffer A) to yield a total volume of
pentanediol (MPD), 4% PEG 8000, and 1% sodium cacodylate
with protein at 20 mg/mL mixed with mother liquor in a 1:1
ratio at 4 °C. All crystals were cryoprotected with mother
liquor supplemented with 30% glycerol and then vitrified in
liquid nitrogen.
2
4
∼
15 mL. The cell suspension was chilled on ice for 15 min,
after which the cells were disrupted by sonication for 3 min at
0% duty cycle/50% output using a Vibra-Cell sonicator model
5
VC250B (Sonics & Materials, Inc., Danbury, CT). Unbroken
cells and debris were removed by centrifugation (45 min,
Data Collection, Processing, Structure Determina-
tion, and Refinement. X-ray diffraction data for N1 and N2
crystals were collected at Advanced Photon Source (APS)
beamline 23-ID-B. Diffraction data were indexed, integrated,
and scaled using HKL2000. The phase was determined for N1
1
8000g). The clear supernatant was subsequently applied to a
DEAE Sepharose column (∼15 mL bed volume), which had
previously been equilibrated in buffer A. The column was
washed with buffer A (3 × 15 mL), and retained proteins were
subsequently eluted with a linear gradient of buffer A made
20
and N2 using PHASER from the PHENIX software suite.
Both structures were determined by molecular replacement
using as search models Cg10062 [Protein Data Bank (PDB)
entry 3N4G] for N1 and the 4-OT homologue TomN (PDB
5
1
00 mM in NaCl. Typically, both N2 and N1 eluted around
50−200 mM NaCl.
The fractions from the DEAE Sepharose column containing
21
entry 3RY0) for N2. Structure refinement was carried out
using PHENIX Refine. The TLS parameter was included in the
refinement of all structures. The final structures were evaluated
the target protein were pooled and made 1 M in (NH ) SO
4
2
4
by the addition of the appropriate volume of a 3.2 M
NH ) SO stock solution in buffer A. The sample was
2
2
(
during and after refinement using Molprobity. Data
collection and refinement statistics for all structures are
summarized in Table 1. Figures were prepared using PyMol
(The PyMOL Molecular Graphics system, version 1.8,
4
2
4
incubated on ice for 2 h, after which precipitates were removed
by centrifugation (20 min, 18000g). The clear supernatant was
then loaded onto a Phenyl Sepharose column that had
previously been equilibrated in buffer A, made 1 M in
23
Schrodinger, LLC).
(
3
NH ) SO . Unbound proteins were removed by washing with
Steady-State Kinetics. The enol−keto tautomerization of
2-hydroxymuconate (2-HM) and phenylenolpyruvate (PP)
was assayed at 22 °C in 20 mM Na HPO buffer (pH 7.3)
4
2
4
column volumes of buffer A, made 1 M in (NH ) SO .
4 2 4
Retained proteins were subsequently eluted with a linear
gradient of buffer A. N2 and N1 typically eluted around 600
and 300 mM (NH ) SO , respectively.
2
4
using various enzyme concentrations (0.6−2.25 μM) depend-
21,24
ing on the catalytic efficiency of each enzyme.
Stock
4
2
4
The fractions containing the target protein were pooled and
concentrated to a volume of ∼3 mL using an Amicon
concentrator equipped with a 3 kDa cutoff membrane filter.
This sample was subsequently applied to a size-exclusion
column (Bio-Gel P60 beads, 500 mL bed volume), which had
previously been equilibrated in buffer A, and proteins were
eluted with buffer A (0.5 mL/min) at 22 °C. The protein-
containing fractions (5 mL) were analyzed by SDS−PAGE,
and those containing near-homogeneous target protein were
pooled and concentrated to ∼20 mg/mL using an Amicon
concentrator. Aliquots were flash-frozen in liquid nitrogen and
stored at −80 °C until further use.
Light Scattering Experiments. The light scattering
experiments were carried out at 25 °C on a DAWN
HELEOS-II multiangle light scattering photometer with an
Optilab T-rex refractometer detector and a Wyatt QELS
dynamic light scattering detector (MALS-QELS system)
solutions of 2-HM (50 mM) and PP (100 mM) were prepared
by dissolving the appropriate amount of the free acid in
absolute ethanol. The ketonization of 2-HM by N2 was
monitored by following the depletion of the enol form at 330
−
1
−1
nm (ε = 934 M cm ) using substrate concentrations
ranging from 26 to 300 μM. The ketonization of PP by both
N2 and N1 was monitored by following the depletion of the
−
1
−1
enol form at 306 nm (ε = 3675 M cm ) using substrate
concentrations ranging from 170 to 1030 μM. An appropriate
quantity of enzyme [from an ∼20 mg/mL stock solution in 10
mM Na HPO buffer (pH 7.3)] was diluted in sodium
2
4
phosphate buffer (780 μL in a 2 mm path length quartz
cuvette), and the assay was initiated by the addition of a small
aliquot (3−25 μL) of the substrate (either 2-HM or PP) from
a stock solution using a positive displacement pipet. At all
substrate concentrations, the non-enzymatic rate was sub-
tracted from the enzymatic rate of ketonization.
(
Wyatt Technology, Santa Barbara, CA), as described
The hydration of propiolate, 2,3-butadienoate, and 2-oxo-3-
pentynoate (2-OP) (Scheme 1) by both N2 and N1, as well as
the hydrolytic dehalogenation of cis-3-chloroacrylate by N1,
was monitored according to procedures published else-
1
9
elsewhere. Samples were delivered through a TSK-GEL
G300PWXL size-exclusion column (7.8 mm × 300 mm, pore
size of 300 Å) (Tosoh Bioscience LLC, King of Prussia, PA)
connected to a Shimadzu LC-20AD HLPC system (model
WTC-030S5). The solvent was 10 mM NaH PO buffer (pH
25−27
where,
with the following modifications. The hydration
2
4
7
.3), and the flow rate was 0.4 mL/min. Molar mass moments
Scheme 1. Compounds Used in This Work
in grams per mole were determined with 20 μL samples at
concentrations of 25 and 2.5 μM. Molar masses, peak
concentrations, and hydrodynamic radii were determined
with Astra 6 software (Wyatt Technology).
Crystallization. Initial crystallization conditions for N1 and
N2 were identified using sparse-matrix screening with a
Phoenix crystallization robotic system (Art Robbins Instru-
ments). After systematic optimization of the hits, reproducible
N1 crystals grew under the condition consisting of 100 mM
Tris buffer (pH 7.0), 35% PEG 3550, and 200 mM Li SO
2
4
with protein at 70 mg/mL at 4 °C. N2 crystals grew in vapor
diffusion sitting drops under conditions of 40% 2-methyl-2,4-
1
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−
1
of 2,3-butadienoate was monitored at 230 nm (ε = 790 M
2 or linker 1. Of the characterized proteins, N2 sequences are
connected only to those of N1.
−
1
−1
−1
cm ) and 250 nm (ε = 250 M cm ) for N1 and N2,
respectively.
NMR Analysis. The protocol for monitoring the reaction of
N1 or N2 with 2,3-butadienoate (allene), 2-butynoate, and
propiolate by H NMR spectroscopy followed that described
Accordingly, an amount of each compound (4
mg) was added to 100 mM Na HPO buffer (pH ∼9.2)
containing 30 μL of dimethyl sulfoxide-d (DMSO-d ) (final
Purification of N2 and N1 and MS and Light
Scattering Analysis. Both N1 and N2 were overproduced
in E. coli BL21(DE3) and purified to near homogeneity. The
typical yield was ∼40 mg for N1 and ∼100 mg for N2 (per liter
of culture). MS analysis of the purified N1 showed a single
major signal corresponding to a monoisotopic mass of 16104
Da (calcd 16235 Da) in the reconstructed mass spectrum. MS
of the purified N2 showed a single major signal corresponding
to a monoisotopic mass of 7010 Da (calcd 7141 Da) in the
reconstructed mass spectrum. The mass difference of 131 Da
between the theoretical and observed mass indicates that the
translation-initiating methionine was post-translationally re-
moved, resulting in the mature and catalytically active proteins
1
2
5,26
elsewhere.
2
4
6
6
total volume of 600 μL). The final pH of the solution was
adjusted to 8.0. Subsequently, an aliquot of N1 or N2 was
added to the mixture such that the final amount of protein was
0
.5 mg. For overnight NMR runs (i.e., 18 h), product
formation was determined using a Bruker AVANCE III 500
MHz spectrometer. For shorter NMR runs, product formation
was monitored every 3 min for 10 intervals on a Varian (Palo
Alto, CA) DirectDrive 600 MHz spectrometer. The initial
spectrum was recorded 3 min after mixing. DMSO-d was used
as the lock signal and for standardization of the chemical shifts
at 2.49 ppm). The chemical shifts for the products are
The approximate amount of product
in the mixture was determined by integration, as reported
elsewhere. NMR signals were analyzed using SpinWorks
version 3.1.6 (Copyright 2009 Kirk Marat, University of
Manitoba, Winnipeg, MB).
Protein Labeling Studies. The monoisotopic masses of
native, unmodified N2 and N1 were determined by MS
analysis using protein samples diluted to ∼25 μM in 10 mM
NaH PO buffer (pH 7.3) from ∼20 mg/mL stock
(
144 amino acids for N1 and 64 amino acids for N2) with an
N-terminal proline. Light scattering analysis of N1 showed a
single peak (100% mass fraction) with a molar mass of 48700
Da (±3.7%). This species is likely the trimer form of N1 (calcd
6
4
8312 Da). Light scattering analysis of N2 showed a single
(
7,26,27
peak (100% mass fraction) with a molar mass of 42720 Da
reported elsewhere.
(
4
±3.7%). This species is likely the hexamer form of N2 (calcd
2
6
2060 Da).
Sequence and Structural Analyses of N1 and N2. The
conserved residues in N2 and N1 were aligned with those of 4-
oxalocrotonate tautomerase (4-OT), fused 4-OT, linker 2,
linker 1, CgX, and cis-3-chloroacrylic acid dehalogenase (cis-
CaaD) (Figure 2). The sequence of N1 aligns well with the
2
4
concentrations. Samples to determine the potential covalent
modification of N2 and N1 by 2-oxo-3-pentynoate (2-OP)
were prepared as follows. A stock solution of 2-OP (60 mM)
was prepared by dissolving 4 mg of the compound in 100 mM
NaH PO buffer (600 μL, pH ∼9) and adjusting the final pH
2
4
to 7.0. Potential labeling reactions of 2-OP were initiated by
the addition of ∼1 mg of protein (diluted from an ∼20 mg/mL
stock solution) to 1 mL of 10 mM NaH PO buffer (pH 7.3)
2
4
Figure 2. Alignment of conserved N1 and N2 sequence motifs with
containing 2-OP (6 mM). After incubation for 1 h at 22 °C,
the structure-based alignment of previously characterized 4-OT
NaBH (from a 1 M stock solution in deionized water) was
1
4
superfamily members as shown in Figures 3 and 4. The 4-OT
added to a final concentration of 25 mM, and the resulting
reaction mixture was incubated at 22 °C for 3 h. Subsequently,
the reaction mixtures were buffer-exchanged into 10 mM
NaH PO buffer (pH 7.3) using a PD-10 column. The elution
subgroup members are colored blue (with conserved residues boxed
in blue), and those from the cis-CaaD subgroup are colored red (with
conserved residues boxed in red). The numbers at the top correspond
to the residue numbers in N2, and the numbers at the bottom
correspond to those in N1.
2
4
fractions containing protein were pooled, flash-frozen in liquid
N , and stored at −80 °C until MS analysis.
2
RESULTS AND DISCUSSION
■
conserved residues in the other members of the cis-CaaD
subgroup (boxed in red). In N2, Arg-8 replaces Leu-8 (and Ile
in fused 4-OT and linker 2) but aligns with Arg-70 (using the
N1 numbering system) in linker 1, CgX, cis-CaaD, and N1
(boxed in red). In addition, Tyr-39 in N2 replaces the
conserved Arg (Arg-39 in 4-OT) in the 4-OT subgroup. While
detailed sequence analysis (Figure 1 and ref 1) clearly places
N2 in the 4-OT subgroup, the motif alignment shown in
Figure 2 suggests it also has “cis-CaaD-like” properties,
particularly at the key position Arg-8 of the 4-OT subgroup
(position Arg-70 of the cis-CaaD subgroup).
Discovery of N2 and N1 and Sequence Analysis.
Linker proteins N1 and N2 were discovered in the course of
the global analysis of the TSF where their representative nodes
were connected through a representative linker edge between
the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and 4-
1
oxalocrotonate tautomerase (4-OT) subgroups. In that work,
sequences from the representative nodes within the individual
subgroups show a connection trajectory in which N1
sequences are connected to sequences comprising linker 1,
and to those of other representative nodes of the cis-CaaD
homologue (CgX), and cis-CaaD, but are not connected to
those of linker 2 (in the 4-OT subgroup) (Figure 1).
Sequences of the N2 representative node are not directly
connected to those comprising canonical 4-OT or fused 4-OT
representative nodes. Nor are they connected to those of linker
The structure of N1 was determined by X-ray crystallog-
raphy at a resolution of 2.45 Å. One trimer exists in each
asymmetric unit in space group P2 2 2 , with 120° of rotation
1
1 1
for each monomer. The crystal structure of N2 diffracts to 1.65
Å in space group R32. With two monomers in each asymmetric
1
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Figure 3. Crystal structures of N1. The overall structures are shown in ribbon diagrams with conserved residues shown as sticks. (A) Overall
structure of N1 with putative active site residues labeled. The secondary structure is shown as a ribbon diagram with the β-strand colored light blue
and α-helices colored lime green. (B) Superimposition of N1, CgX, and cis-CaaD. N1 is colored using the same color scheme as in A; however,
CgX is colored lilac, and cis-CaaD is colored pink.
unit, crystallographic symmetry generates the functional unit of
a homohexamer with six subunits.
from the prolyl nitrogen and 3.2 Å from the hydroxyl group of
Tyr-39 and could play a role in hydration.
N1 is in the cis-CaaD subgroup and has the same set of six
active set residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103,
and Glu-114) as those found in cis-3-chloroacrylic acid
Kinetic Properties of N1 and N2. N1 and N2 were
screened with seven known TSF substrates (Scheme 1) to
1
,21,24
assess their 4-OT-like and cis-CaaD-like properties.
The
28
dehalogenase (cis-CaaD) and the cis-CaaD homologue,
kinetic parameters are listed in Table 2. Phenylenolpyruvate
(PP) and 2-hydroxymuconate (2-HM) assess tautomerase
activity (a 4-OT-like property), whereas cis-3-chloroacrylate,
propiolate, 2,3-butadienoate, 2-butynoate, and 2-OP uncover
2
6,29
CgX
(Figures 2 and 3A,B) except that linker 1 shows an
alanine in place of the tyrosine at position 103. Importantly,
the residues are positionally conserved in those structures, and
the active sites are nearly superimposable (Figure 3B). On the
basis of the structural similarities, it is anticipated that N1 will
have activities similar to those of CgX and cis-CaaD using
similar substrates with varying efficiencies. Note that the two
1
hydration potential (a cis-CaaD property). The previous
kinetic analysis of the enzymes linking the 4-OT and cis-CaaD
subgroups showed a loss of 4-OT activity and an increase in
hydration activity that culminates in the cis-CaaD-catalyzed
1
1
arginine residues are to one side of the catalytic Pro-1.
hydrolytic dehalogenation. 4-OT, fused 4-OT, and linker 2
N2 belongs to the 4-oxalocrotonate tautomerase (4-OT)
subgroup. Sequence analysis and structure analysis reveal that
the active site configuration for canonical 4-OT activity is only
partially conserved, where canonical 4-OT activity refers to
that using 2-hydroxymuconate (2-HM), a dicarboxylate
substrate (Schemes 1 and 2). In N2, Arg-11 is conserved but
(
in the 4-OT subgroup) were found to be proficient
6
7
−1 −1
tautomerases using 2-HM (k /K ∼ 10 −10 M s ) or
cat
m
5
6
−1 −1
1
PP (k /K ∼ 10 −10 M s ) (Schemes 1 and 2). Linker
cat
m
1
, CgX, and cis-CaaD (in the cis-CaaD subgroup) show no
detectable 4-OT activity with 2-HM, but a modest tautomerase
activity using PP (k /K ∼ 10 −10 M s ). Analysis of
3
5
−1 −1
1
cat
m
the active sites shows that in the 4-oxalocrotonate tautomerase
subgroup members, Pro-1 is positioned between two arginine
groups (positionally comparable to Arg-11 and Arg-39 in 4-
OT) such that the di- and monocarboxylate substrates can be
processed by 4-OT, fused 4-OT, and linker 2 (Figure 4B).
However, in linker 1, CgX, and cis-CaaD, two arginine groups
Scheme 2. Enzyme-Catalyzed Tautomerization Reactions
(
positionally comparable to Arg-70 and Arg-73 in cis-CaaD)
are to one side of Pro-1 so that 2-HM is not processed, but PP
is (Figure 3B).
Kinetic analysis of N2 shows both substrates [phenyl-
enolpyruvate (PP) and 2-hydroxymuconate (2-HM)] are
processed, but activity with 2-HM is markedly decreased
2
−1 −1
(
k /K ∼ 10 M s ), compared with those of other
cat
m
6 7
characterized 4-OT subgroup members (k /K ∼ 10 −10
cat
m
−
1 −1
1
M
s ). Notably, in N2, Pro-1 is not between two arginine
residues because Arg-39 is replaced with a tyrosine (Figure
4A). Hence, this substitution can account for the decreased
activity of N2 with 2-HM. However, like linker 1, CgX, and cis-
CaaD, two arginines (Arg-8 and Arg-11) are to one side of
Arg-39 is replaced by a tyrosine (Figures 2 and 4A,B).
Moreover, Leu-8 (in 4-OT) is replaced with Arg-8 (Figure
4
B). In 4-OT, the nucleophilic Pro-1 is sandwiched between
two arginine residues (Arg-11 and Arg-39), but in N2, the two
arginine residues are placed to one side of Pro-1 (Figure 4B).
This property is a characteristic of cis-CaaD subgroup
Pro-1 (a cis-CaaD-like property) so that PP activity (kcat/K ∼
m
3
−1 −1
10 M s ) is observed but is also reduced compared to that
of 4-OT. The active site of N1 is similar to those of linker 1,
1
1
members. In addition, a water molecule is located 2.8 Å
CgX, and cis-CaaD where two arginine groups are to one side
1
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Figure 4. Crystal structures of N2. The overall structures are shown as ribbon diagrams with active site residues shown as sticks. (A) Overall
structure of N2 with putative active site residues labeled. The secondary structure is shown as a ribbon diagram with β-strands colored silver and α-
helices colored teal. The active site residues are numbered. (B) Superimposition on N2 and 4-OT. N2 is colored in the same color scheme as in
panel A, but 4-OT is colored violet with the nonconserved residues labeled in italics.
of Pro-1 (Figure 3B). Hence, the monocarboxylate substrate
min, a reaction mixture of N2 and allene consisted of the
unreacted substrate (20.7%), acetoacetate (77.9%), and
acetone (1.4%). The amount of decarboxylated product
could be due to enzyme catalysis. Although a little more
4
−1
−1
(
PP) is processed (k /K ∼ 10
M
s ), but the
cat
m
dicarboxylate substrate (2-HM) is not.
2
N1 shows activity with cis-3-chloroacrylate (k /K ∼ 10 )
cat
m
1
and more significant activity with two other substrates,
propiolate (k /K ∼ 10 ) and 2,3-butadienoate (k /K ∼
1
difficult to categorize, H NMR analysis shows that N2
3
functions mostly as a hydratase with weak decarboxylase
activity.
cat
m
cat
m
3
0 ) (Scheme 3 and Table 2). The first two compounds are
1
processed to malonate semialdehyde (MSA), and the third
compound is converted to acetoacetate (Scheme 3). The
reactions of N1 with propiolate and 2,3-butadienoate were
N1 and N2 also process 2-butynoate, as assessed by H
NMR spectroscopy. After 18 h, a reaction mixture of N1 and 2-
butynoate consisted of acetoacetate (38.7%), acetone (11.1%),
and starting material (50.2%). The amount of decarboxylated
product is larger than that expected for non-enzymatic activity.
After 18 h, a reaction mixture of N2 and 2-butynoate consisted
of acetoacetate (78.6%), acetone (4.8%), and starting material
(16.6%). The amount of decarboxylated product is that
expected for non-enzymatic activity. The reaction of N2 is
clearly faster than that of N1 (using 2-butynoate). The reaction
of CgX with 2-butynoate is comparable, but much faster. After
39 min, the reaction mixture consists of mostly acetoacetate
and a small amount of acetone (2.5%). The amount of acetone
1
examined further by H NMR spectroscopy to determine if
hydration is followed by decarboxylation. (Decarboxylation
cannot be detected by the UV assay.) After 30 min, a reaction
mixture of N1 and propiolate consisted of the unreacted
substrate (83.9%), MSA (0.3%) and its hydrate (1.2%), and
acetaldehyde (6.3%) and its hydrate (8.3%). The amount of
decarboxylated product exceeds that expected for the non-
enzymatic reaction. After 18 h, a reaction mixture of N1 and
2
,3-butadienoate consisted of acetoacetate (97.6%) and
acetone (2.4%). The amount of decarboxylated product is
comparable to that expected for non-enzymatic activity (after
26
exceeds that expected for the non-enzymatic decarboxylation.
1
8 h). Hence, N1 catalyzes hydration/decarboxylation with
The activity of N2 is faster than that of N1 using propiolate
and comparable to that of N1 using the allene, as assessed by
the kinetic parameters and H NMR analysis. N1 and N2 also
propiolate, but only hydration with the 2,3-butadienoate.
These activities (hydration/decarboxylation vs hydration)
parallel those of CgX, but at a much lower efficiency. For CgX,
1
process 2-butyonate to acetoacetate (Scheme 3), as assessed by
1
the reactions with propiolate and 2,3-butadienoate have k /
H NMR analysis. Again, N2 is faster than N1. The significant
cat
5
3
K values of ∼10 (propiolate) and ∼10 (2,3-butadienoate)
hydration activity coupled with the modest tautomerase
activity is typically associated with members of the cis-CaaD
subgroup. Although the overall sequence similarity is
significant enough for N2 to be placed in the 4-OT subgroup,
the presence of cis-CaaD-like machinery at critical active site
positions [two arginine residues to one side of Pro-1 (Figure
4B)] could cause the observed reaction preferences along with
the presence of Tyr-39, which might position a water molecule
to facilitate hydration. We have previously shown that
replacement of Leu-8 with Arg-8 enhances the hydration
m
(Table 2). For the allene, both CgX and N1 show high K
m
values (780 and 3100 μM, respectively), which could be a
function of the rigid and bulky allene system. For CgX, H
NMR analysis shows that both reactions are complete in 3
min: with prolionate, the product is mostly the decarboxylation
product, acetaldehyde (75%) (i.e., hydration/decarboxylation),
and with 2,3-butadienoate, the product is 100% acetoacetate
1
(
i.e., hydration).
N2 also shows mostly hydratase activity with both
4
15
substrates. For propiolate, the k /K is ∼10 , and for 2,3-
activity of 4-OT (using trans-3-CAA). Taken together, these
cat
m
butadienoate, the k /K is ∼67 (also with a high K value of
observations provide experimental evidence for the transitional
properties of N2, as suggested by the SSN analysis that
identifies N2 as a linker protein between the 4-OT and cis-
CaaD subgroups.
Incubation of N1 or N2 with 2-Oxo-3-pentynoate (2-
OP). N1 and N2 were incubated in separate reaction mixtures
cat
m
m
1
6100 μM). The reactions of N2 with propiolate and allene
1
were examined further by H NMR spectroscopy. After 35
min, a reaction mixture of N2 and propiolate consisted of the
unreacted substrate (73%), malonate semialdehyde (8%),
hydrate (19%), and trace amounts of acetaldehyde. After 35
1
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Scheme 3. Enzyme-Catalyzed Reactions with Various
Substrates
with 2-OP at pH 7.3, as described in Experimental Procedures.
After incubation for various amounts of time, the enzymes
were recovered and analyzed by MS. The masses of the major
signals are summarized in Table 3.
Table 3. Masses Corresponding to the Major Signals
Observed in the Reconstructed Spectra of the Incubation
Reaction Mixtures of N1 (native mass of 16104 Da) and N2
(
native mass of 7010 Da) with 2-OP
N1
N2
a
observed mass (Da) ratio
16104 2:1
6217
observed mass (Da)
7010
ratio
2
-OP
1:1:1.5
b
1
7052
7
123
a
b
The ratio represents the relative signal intensity. The +42 Da
adduct is likely an artifact of the MS conditions.
Both N1 and N2 were covalently modified by 2-oxo-3-
pentynoate (2-OP) as reflected by the signals at 16217 and
7
123 Da, respectively, corresponding to an adduct with a mass
of 113 Da (Scheme 4). 2-OP is used as a probe for the pKa
value of the prolyl nitrogen (of Pro-1) on the basis of its
3
,31,32
behavior with different TSF members.
In general, if the
pK is low (<7), the enzyme is irreversibly inactivated by the
a
formation of a covalent bond between the prolyl nitrogen and
14
C-4 of 2-OP (Scheme 4). If the pK is high (>9), the enzyme
a
catalyzes a hydration reaction to form acetopyruvate at pH ∼7
3
,31,32
(
Scheme 3).
a
In 4-OT, the prolyl nitrogen of Pro-1 has a
1
5
5
pK of 6.4, as measured by N NMR spectroscopy, and the
enzyme is irreversibly inhibited by covalent modification of the
14
prolyl nitrogen. For N2, the results parallel those of 4-OT,
suggesting that Pro-1 has a low pK value. Notably, most of the
a
protein is labeled (∼71.4%) and there is no detectable
hydration (see Tables 2 and 3). N1, on the contrary, is in the
1
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Scheme 4. Mechanism for Inactivation by 2-OP and Reduction by NaBH4
cis-CaaD subgroup. The pK of Pro-1 in cis-CaaD is 9.3,
ASSOCIATED CONTENT
Accession Codes
The atomic coordinates and structure factors have been
deposited as Protein Data Bank entries 7M58 for N1 and
M59 for N2. N1 from C. halotolerans YIM 70093 (UniProt
a
■
consistent with its role as a general acid in the dehalogenation
3
,33
reaction.
along with some inactivation. N1 is also modified by 2-OP
∼33.3%), but not as extensively as N2. The N1-catalyzed
Hence, cis-CaaD catalyzes the hydration of 2-OP
3
(
7
hydration of 2-OP could also decrease the amount of 2-OP for
reaction with the prolyl nitrogen (Table 2). Nonetheless, if
these observations can be viewed as rough comparisons of the
entry M1NLA4). N2 from G. bacterium SG8_31 (UniProt
entry A0A0S8FF56).
pK values of the prolines, it suggests that they are different and
AUTHOR INFORMATION
Corresponding Author
a
■
the pK of Pro-1 in N1 is higher than the pK of Pro-1 in N2.
a
a
Christian P. Whitman − Division of Chemical Biology and
Medicinal Chemistry, College of Pharmacy and Institute for
Cellular and Molecular Biology, The University of Texas at
Austin, Austin, Texas 78712, United States; orcid.org/
CONCLUSIONS
■
Two previously identified linker nodes (N1 and N2) in the
SSN that connect the cis-3-chloroacrylic acid dehalogenase
0
000-0002-8231-2483; Phone: (512) 471-6198;
Email: whitman@austin.utexas.edu; Fax: (512) 232-2606
(
cis-CaaD) and 4-oxalocrotonate tautomerase (4-OT) sub-
1
groups were subjected to sequence, structure, and kinetic
characterization. Sequence analysis places N1 in the cis-CaaD
subgroup and N2 in the 4-OT subgroup. N1 functions like the
other characterized cis-CaaD subgroup members with both
tautomerase (using a monocarboxylate, but not a dicarboxylate
substrate) and hydratase activities (using cis-CAA, allene, and
acetylene substrates). The active site of N1 mirrors those of
the other cis-CaaD subgroup members and is consistent with
the kinetic profile. N2, however, does not behave like other
characterized 4-OT subgroup members but functions in a cis-
CaaD-like manner. It has significantly reduced tautomerase
activity (using mono- and dicarboxylate substrates) and
enhanced hydratase activity (using allene and acetylene
substrates). Structural analysis shows two substitutions that
might convey cis-CaaD-like behavior. Leu-8 (in 4-OT) is
replaced by an arginine, and Arg-39 is replaced by a tyrosine.
As a result, Pro-1 is now not between two arginine residues,
but on one side of them. This is a common feature of cis-CaaD
subgroup members. Structural analysis further suggests that
Tyr-39 might position a water molecule for fortuitous addition
to the substrate. These active site changes are simple but were
not predictable ones from sequence function patterns defined
by large-scale analysis of more than 11000 TSF members,
underscoring the difficulty of functional assignment of
proteins.
Authors
Bert-Jan Baas − Division of Chemical Biology and Medicinal
Chemistry, College of Pharmacy, The University of Texas at
Austin, Austin, Texas 78712, United States
Brenda P. Medellin − Department of Molecular Biosciences,
The University of Texas at Austin, Austin, Texas 78712,
United States
Jake A. LeVieux − Department of Molecular Biosciences, The
University of Texas at Austin, Austin, Texas 78712, United
States
Kaci Erwin − Division of Chemical Biology and Medicinal
Chemistry, College of Pharmacy, The University of Texas at
Austin, Austin, Texas 78712, United States
Emily B. Lancaster − Division of Chemical Biology and
Medicinal Chemistry, College of Pharmacy, The University of
Texas at Austin, Austin, Texas 78712, United States
William H. Johnson, Jr. − Division of Chemical Biology and
Medicinal Chemistry, College of Pharmacy, The University of
Texas at Austin, Austin, Texas 78712, United States
Tamer S. Kaoud − Division of Chemical Biology and
Medicinal Chemistry, College of Pharmacy, The University of
Texas at Austin, Austin, Texas 78712, United States
R. Yvette Moreno − Department of Molecular Biosciences,
The University of Texas at Austin, Austin, Texas 78712,
United States
1
784
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Marieke de Ruijter − Division of Chemical Biology and
Medicinal Chemistry, College of Pharmacy, The University of
Texas at Austin, Austin, Texas 78712, United States
Patricia C. Babbitt − Departments of Bioengineering and
Therapeutic Sciences, Department of Pharmaceutical
Chemistry, and Quantitative Biosciences Institute, University
of California, San Francisco, San Francisco, California
sulfate−polyacrylamide gel electrophoresis; trans-3-CAA, trans-
3-chloroacrylic acid dehalogenase; TLS, translation-libration-
screw-rotation; TSF, tautomerase superfamily; UV, ultraviolet.
REFERENCES
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(
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(
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OD021508−01 (Bruker AVANCE III 500 MHz spectrom-
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(
ACKNOWLEDGMENTS
■
P. (2003) Reactions of trans-3-chloroacrylic acid dehalogenase with
acetylene substrates: consequences of and evidence for a hydration
reaction. Biochemistry 42, 8762−8773.
The protein mass spectrometry analysis was conducted in the
Institute for Cellular and Molecular Biology Protein and
Metabolite Analysis Facility at The University of Texas at
Austin. The authors thank Steve D. Sorey (Department of
Chemistry, The University of Texas at Austin) for his expert
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assistance in the acquisition of the H NMR spectra.
Instrumentation and technical assistance for this work were
provided by the Macromolecular Crystallography Facility, with
financial support from the College of Natural Sciences, the
Office of the Executive Vice President and Provost, and the
Institute for Cellular and Molecular Biology at The University
of Texas at Austin. The Berkeley Center for Structural Biology
is supported in part by the National Institutes of Health,
National Institute of General Medical Sciences, and the
Howard Hughes Medical Institute. The Advanced Light
Source is supported by the Director, Office of Science, Office
of Basic Energy Sciences, of the U.S. Department of Energy
under Contract DE-AC02-05CH11231.
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ABBREVIATIONS
APS, Advanced Photon Source; CHMI, 5-(carboxymethyl)-2-
hydroxymuconate isomerase; cis-CaaD, CC, or cis-3-CAA, cis-
■
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3
-chloroacrylic acid dehalogenase; CgX, cis-CaaD homologue
designated Cg10062 from Corynebacterium glutamicum; DEAE,
diethylaminoethyl; DMSO, dimethyl sulfoxide; MS, mass
spectrometry or mass spectrometric; F4-OT, fused 4-
oxalocrotonate tautomerase; 2-HM, 2-hydroxymuconate; Kn,
kanamycin; LB, Luria-Bertani; MPD, 2-methyl-2,4-pentane-
diol; MIF, macrophage migration inhibitory factor; MSA,
malonate semialdehyde; MSAD, malonate semialdehyde
decarboxylase; MR, molecular replacement; MW, molecular
weight; MAD, multiwavelength anomalous dispersion; NMR,
nuclear magnetic resonance; 4-OT, 4-oxalocrotonate tauto-
merase; PP, phenylenolpyruvate; PEG, polyethylene glycol;
PDB, Protein Data Bank; 2-OP, 2-oxo-3-pentynoate; SSN,
sequence similarity network; SDS−PAGE, sodium dodecyl
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