Biochemistry
Article
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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Biochemistry 2021, 60, 1776−1786