E. coli RutC is a 3-AA deaminase
ꢀ
NADPH consumed was determined by the decreased absor-
C with constant shaking. Growth was quantified by moni-
bance at 340 nm using the molar absorptivity of NADPH toring the absorbance at 650 nm.
−
1
−1
(
6220 M cm ). NADPH (0.8 mM) was added (9), and its
consumption over time was determined (Fig. S1). Rates of Molecular biology
NADPH oxidation versus ureidoacrylate concentration were
R104A
Wildtype rutC and the allele encoding RutC
(nucleo-
graphed in Prism, version 7 (GraphPad) to define apparent
tides 310–312 were changed from CGA to GCA) were PCR
amplified from pDM1677 and pDM1668, respectively, using
primers PR1513 and PR1514 for cloning into vector pCV1 (21)
and primers PR42 and PR43 for cloning into pET28b-
kinetic parameters.
Assay for 2-AA deaminase activity
A coupled-enzyme assay (final volume of 300 μl) consisting SAPKOCH. PCR products and vectors were digested with
of CdsH (0.19 μM), lactate dehydrogenase (5 U), NADH BspQI and ligated together using technique described by
(0.25 mM), pyridoxal-5-phosphate (30 μM), RidA (0.3 μM) or Galloway et al. (22). The partial in-frame deletion of rutD
RutC (0.3 μM), and L-cysteine (L-cys) in Trizma base buffer found in pDM1661 was generated using primers PR1511 and
ꢀ
(
100 mM, pH 8.0 at 23 C) was used to determine 2-AA PR1512 to PCR amplify the regions of pDM16568 upstream
deaminase activity (20). Assay was performed in technical and downstream of the portion of rutD targeted for deletion
triplicate in a quartz microplate and initiated by addition of (nucleotides 46–750). The blunt ends of the PCR product were
varying concentrations of L-cys. Using Spectramax Plus 384 ligated with plasmid to generate pDM1661. In all cases, liga-
microplate reader and Softmax Pro 6.2 software, the rate of tion mixtures were transformed into DH5α, and transformants
pyruvate formation was quantified by measuring the change in were selected for the appropriate antibiotic resistance. Plas-
−
1
−1
absorbance of NADH at 340 nm (6200 M cm ) from 20 to mids were verified for sequence by Eurofins Genomics.
0 s. Prism, version 7, was used to graph data and determine
5
statistical significance by one-way ANOVA followed by Protein overexpression and purification
Tukey’s post hoc test.
YdfG
An LB chloramphenicol culture of BL21 carrying pCA24N-
Assay for iminoacid deaminase activity
ydfG (1.5 ml) was used to inoculate 1.5 l of LB containing
A coupled-enzyme assay (final volume of 100 μl) consisting of
bovine liver catalase (1 μg), L-amino acid oxidase (1 μg), neutral-
ized semicarbazide (10 mM), RidA (10 μM) or RutC (10 μM), and
various L-amino acids (10 mM) in potassium pyrophosphate
ꢀ
chloramphenicol. Cells were grown at 37 C with shaking (150
RPM). When an absorbance at 600 nm reached ꢁ0.5, cells were
induced by adding IPTG to 100 μM. Cultures were grown for
ꢀ
1
7 h at 15 C (150 RPM) and harvested at 4000 RPM for 10 min
ꢀ
buffer (50 mM, pH 8.7 at 23 C) was performed to determine
ꢀ
ꢀ
at 4 C. The resulting cell pellet was stored at –80 C until use.
iminoacid deaminase activity (8). Reactions were initiated with
addition of L-aminoacids, andthechangeinabsorbanceat248nm
was monitored using a Spectramax Plus 384 microplate reader
and Softmax Pro 6.2 software. The assay was performed in tech-
nical triplicate in a quartz plate. Using the molar absorptivity of
Cells were thawed and resuspended (5 ml/g) in bind buffer
potassium phosphate at pH 7.4 [20 mM], NaCl [300 mM], and
(
glycerol [10%]) containing PMSF (1 mM), DNase (20 U/ml),
and lysozyme (1 mg/ml). Cells were lysed using OneShot cell
ꢀ
disruptor at 20 psi. Lysate was cleared at 40,000g, 4 C for
−1
−1
semicarbazone at 248 nm (10,300 M cm ) and the change in
absorbance at 248 nm from 0 to 10 min, the rate of semicarbazone
formation was calculated. Prism, version 7, was used to plot data
and determine statistical significance by one-way ANOVA fol-
lowed by Tukey’s post hoc test.
4
5 min. Cleared supernatant was loaded onto Ni-agarose
column and washed with bind buffer and then wash buffer
potassium phosphate at pH 7.4 [20 mM], imidazole [25 mM],
(
NaCl [300 mM], and glycerol [10%]). Protein was eluted with
potassium phosphate buffer (20 mM, pH 7.4) containing
imidazole (250 mM), NaCl (300 mM), and glycerol (10%).
Elution fractions were collected and visualized on SDS-PAGE
gel. Fractions containing target protein were combined and
dialyzed in potassium phosphate buffer (20 mM, pH 7.4)
containing NaCl (300 mM) and glycerol (10%). Buffers were
changed three times reducing NaCl concentration stepwise
reaching a final buffer of potassium phosphate (20 mM, pH
Growth studies
Isolated colonies of E. coli were patched in biological trip-
licate on LB agar plates containing chloramphenicol and
ꢀ
incubated at 37 C for 14 to 16 h. Patched plates were replica
printed onto NCN medium and NCE agar plates containing
uridine (0.5 mM), MgSO4 (1 mM), and glycerol (0.4%).
7
.4) containing NaCl (100 mM) and glycerol (10%). Protein
Replica-printed plates were incubated at room temperature
ꢀ
ꢀ
was flash frozen in liquid nitrogen and stored at −80 C.
Protein was >95% pure (Fig. S3).
(
23 C) for 2 days.
S. enterica growth studies were performed in biological
triplicate. Inoculum was prepared by incubating 1 ml nutrient
broth cultures containing ampicillin at 37 C with shaking for
ꢀ
RutC
1
6 to 18 h. Cells were pelleted and resuspended in 1 ml NaCl
One and a half liter of super broth containing kanamycin
solution (8.5 g/l). Resuspended cells were used to inoculate was inoculated with BL21/pET28b-rutC, and cultures were
ꢀ
NCE media in a microwell plate (2% inoculum). The plate was incubated at 37 C 150 RPM until an absorbance at 350 nm
incubated in a Biotek EL808 ultra microplate reader set to 37 reached ꢁ0.6 to 0.8. Expression was induced with the
J. Biol. Chem. (2021) 296 100651 7