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K. KINO et al.
method of Laemmli.17) The N-terminal amino acid sequence of the
Materials and Methods
purified enzyme was determined by Kyowa Hakko Kogyo (Tokyo).
The molecular mass was determined with a HiLoad 16/60 Superdex
200 pg column (GE Healthcare). The column was equilibrated with
50 mM Tris–HCl buffer (pH 8.0) containing 0.15 M NaCl, and the
protein was eluted with the same buffer at a flow rate of 0.5 ml/min.
Materials. Bacillus subtilis NBRC3134 (= ATCC6633) was from
the NITE Biological Resource Center (Chiba, Japan). pUC19 vector
was purchased from Nippon Gene (Tokyo). Escherichia coli Rosetta
(DE3) and pET30Xa/LIC Vector Kits were from Merck (Darmstadt,
Germany). A HindIII cassette and a PstI cassette were from Takara Bio
(Shiga, Japan). Hydroxylammonium chloride (iron free, for iron
analysis) was from Kanto Chemical (Tokyo). All other chemicals used
are commercially available and were of chemically pure grade.
Cloning of the gene encoding the purified enzyme. DNA manipu-
lations were performed according to the method of Sambrook et al.,
with minor modifications.18) To obtain a DNA sequence of the purified
enzyme, the cassette PCR method was used.19) Genomic DNA of
B. subtilis NBRC3134 was digested with HindIII or PstI, and these
DNA fragments were ligated to the HindIII and the PstI cassettes
respectively. The resulting DNA fragments were used as templates of
PCR amplification, and the following primer-sets were used: C1–P1
and C1–P2 toward the HindIII fragment, and C1–P3 toward the PstI
fragment (Table 1). The PCR fragments were digested with HindIII or
PstI and ligated to pUC19 vector. The resulting plasmid was used for
sequence analysis. The DNA fragment amplified using C1–P1 was
sequenced using primers V1, V2, S1, and S2; the DNA fragment
amplified using C1–P2 was sequenced using primers V1, S3, and S4;
and the DNA fragment amplified using C1–P3 was sequenced using
primers V1, S5, and S6.
Cultivation of B. subtilis NBRC3134 and preparation of cell-free
extract. NBRC No. 802 medium (1% polypeptone, 0.2% yeast extract,
.
0.1% MgSO4 7H2O) and Production medium (0.1% L-asparagine,
0.5% L-glutamate, 2% mannitol, 0.1% KH2PO4, 0.05% MgSO4 7H2O,
.
.
.
0.05% KCl, 0.000015% FeSO4 7H2O, 0.000016% ZnSO4 7H2O,
.
0.0005% MnSO4 5H2O; pH 6.0, adjusted with NaOH) were pre-
pared.12) First, B. subtilis NBRC3134 was cultivated in NBRC No. 802
medium at 30 ꢀC with shaking at 120 rpm (overnight). Next, cultivated
cells were transferred to Production medium and cultivated for 48 h at
27 ꢀC with shaking at 120 rpm. The cells were harvested by
centrifugation (4;160 ꢁ g, 10 min, 4 ꢀC), resuspended in 50 mM Tris–
HCl buffer (pH 8.0), and then disrupted by sonication at 4 ꢀC. Cellular
debris was removed by centrifugation (20;000 ꢁ g, 30 min, 4 ꢀC), and
the supernatant was collected as cell-free extract.
Overexpression of rizA and characterization of RizA. The gene
encoding the purified enzyme was designated rizA (DDBJ accession
no. AB437349). rizA was amplified from genomic DNA of B. subtilis
NBRC3134 by PCR using primers R1 and R2 (Table 1). The PCR
fragment was ligated into the pET30Xa/LIC vector according to the
protocol that came with the pET30Xa/LIC Vector Kits. The resulting
plasmid, pBsRzcA, was designed to express the gene with an N-
terminal His-tag sequence under the control of the T7 promoter, and
was introduced into E. coli Rosetta(DE3).
Assay of L-arginine hydroxamate (Arg-NHOH) synthesis activity.
reagent mixture containing 20 mM L-arginine (Arg), 200 mM
A
hydroxylammonium chloride (NH2OH), 30 mM ATP, and 30 mM
.
MgSO4 7H2O in 50 mM Tris–HCl buffer (pH 8.0) was prepared. An
enzyme solution was added to the reagent mixture, and this was
incubated at 30 ꢀC for 1–20 h. To detect Arg-NHOH, the colorimetric
method was used with some modifications, as described below.16) After
the reaction, 150 ml of the reaction mixture was prepared, and 75 ml of
8% trichloroacetic acid and 75 ml of 3.4% FeCl3 (dissolved in 2 N HCl)
were added. The precipitant was then removed by centrifugation
(20;000 ꢁ g, 30 min, 4 ꢀC). The supernatant was collected, and the
absorbance at 490 nm was measured with a microplate reader (Bio Rad
Model 550, Bio-Rad Laboratories, Hercules, CA). To determine
enzyme activity, HPLC analysis was performed, and one unit (1 U) of
enzyme activity was defined as the amount of enzyme that produces
1 mmol of Arg-NHOH per min. The protein concentration was
determined by the Bradford method, with bovine serum albumin as
the standard.
First, recombinant E. coli cells were cultivated in 3 ml of Luria-
Bertani medium (1% bacto tryptone, 0.5% yeast extract, 1% NaCl) that
contained 25 mg/ml of kanamycin and 25 mg/ml of chloramphenicol
(final concentrations) at 37 ꢀC with shaking at 160 rpm (overnight).
Next, cultivated cells were transferred to 100 ml of fresh Luria-Bertani
medium that contained 25 mg/ml of kanamycin and 25 mg/ml of
chloramphenicol (final concentrations) and were cultivated for 2 h at
37 ꢀC with shaking at 120 rpm. Isopropyl-ꢁ-D-thiogalactopyranoside
(final concentration, 0.1 mM) was then added, and cultivation was
continued for 18 h at 25 ꢀC with shaking at 120 rpm. The cells were
harvested by centrifugation (4;160 ꢁ g, 10 min, 4 ꢀC), resuspended in
50 mM Tris–HCl buffer (pH 8.0), and then disrupted by sonication at
4 ꢀC. Cellular debris was removed by centrifugation (20;000 ꢁ g,
30 min, 4 ꢀC), and the supernatant was collected and purified with a
Ni-affinity column, HisTrap HP (GE Healthcare). In addition, the
active fraction was desalted with a PD-10 column (GE Healthcare)
equilibrated with 50 mM Tris–HCl buffer (pH 8.0).
Enzyme purification and protein analyses. All purification proce-
dures were performed at 4 ꢀC or on ice, and purification was carried out
by detecting Arg-NHOH synthesis activity. First, ammonium sulfate,
(NH4)2SO4, was added to a cell-free extract up to 30% saturation.
After being stirred for 1 h, the suspension was centrifuged (20;000 ꢁ g,
60 min, 4 ꢀC), and the supernatant was brought to 60% saturation with
(NH4)2SO4. The resulting precipitant (30–60% fraction) was collected
by centrifugation (20;000 ꢁ g, 60 min, 4 ꢀC), and was dissolved in
50 mM Tris–HCl buffer (pH 8.0) containing 0.7 M (NH4)2SO4. After
centrifugation, the supernatant was loaded onto a HiPrep 16/10 Butyl
FF column (GE Healthcare, Buckinghamshire, UK) previously
equilibrated with 50 mM Tris–HCl buffer (pH 8.0) containing 0.7 M
(NH4)2SO4, and the column was washed with the same buffer. The
enzyme was eluted with a linear gradient of 0.7–0 M (NH4)2SO4 at a
flow rate of 2 ml/min. The active fractions were collected and dialyzed
against 50 mM Tris–HCl buffer (pH 8.0). Next, the resulting solution
was loaded onto a Mono Q 5/50 GL column (GE Healthcare)
previously equilibrated with 50 mM Tris–HCl buffer (pH 8.0), and the
column was washed with the same buffer. The enzyme was eluted with
a linear gradient of 0–0.7 M NaCl at a flow rate of 1 ml/min. The active
fractions were collected and concentrated by ultrafiltration. Finally, the
enzyme solution was loaded onto a HiLoad 16/60 Superdex 200 pg
column (GE Healthcare) previously equilibrated with 50 mM Tris–HCl
buffer (pH 8.0) containing 0.15 M NaCl. The enzyme was eluted with
the same buffer at a flow rate of 0.5 ml/min, and the active fractions
were collected and concentrated by ultrafiltration.
The L-amino acid ligase activity of recombinant RizA was assayed
as follows unless otherwise specified: The standard reaction mixture
(total volume, 0.2 ml) contained 0.1 mg/ml of purified His-tagged
.
RizA, 12.5 mM ATP, 12.5 mM MgSO4 7H2O, and 12.5 mM substrate(s)
in 50 mM Tris–HCl buffer (pH 8.0). The reaction was performed at
30 ꢀC for 18 h. To detect peptide synthesis activity, the amount of
phosphate released by the reaction was determined with a Determiner
L IP kit according to the manufacturer’s protocol (Kyowa Medex,
Tokyo). To confirm peptide synthesis in detail, reaction mixtures were
analyzed by MALDI-TOFMS, LC-ESI-MS, HPLC, or NMR. When
NMR analysis was performed, 50 mM potassium phosphate buffer
(pH 8.0) was used for the reaction.
To investigate substrate specificity, every combination of one or
two amino acids selected from the following amino acids was
examined: L-arginine (Arg), L-lysine (Lys), L-histidine (His), L-
glutamine (Gln), L-asparagine (Asn), L-glutamate (Glu), L-aspartate
(Asp), L-alanine (Ala), L-serine (Ser), L-threonine (Thr), glycine (Gly),
L-proline (Pro), L-valine (Val), L-leucine (Leu), L-isoleucine (Ile), L-
methionine (Met), L-cysteine (Cys), L-phenylalanine (Phe), L-trypto-
phan (Trp), and L-tyrosine (Tyr). DL-2-Amino-5-phosphonopentanoic
acid (APV) was used as a structural analog of L-2-amino-5-phosphono-
3-cis-pentenoic acid (APPA), and a reaction with Arg plus APV
was conducted. To test reactivity with D-amino acids, reactions with
To confirm protein purity, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS–PAGE) was performed according to the