W. Song et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
10 mmol maltose in 50 mM Tris$HCl pH 7.5. The DzCPR supernatant
was loaded on a Ni-NTA agarose column and eluted with 50 mM
Tris-HCl buffer containing 250 mM imidazole. The recombinant
proteins were detected via 10% (w/v) sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE). Protein concen-
tration was assessed using the Bradford assay and the recombinant
proteins were stored at ꢂ20 ꢀC.
assembled into full-length cDNAs of 1927 and 2475 nucleotides,
respectively. The SE and CPR coding regions contained 1542 and
2076 bp, encoding 514 and 692 amino acids, respectively.
The predicted molecular mass and theoretical isoelectric point
(pI) of DzSE were 55.7 kDa and 8.83, respectively. Amino acid
sequence analysis revealed that DzSE belongs to the PLN02985
super family and has a putative FAD-binding domain, which is an
essential site for enzyme catalytic activity. Another major func-
tional domain is an NAD (P)-binding site which is part of a
Rossmann-fold to bind FAD and NADPþ (fig. S1). The sequence
NMRHPLTGGGMTV was conserved in the alignment and binds
squalene as a substrate. Transmembrane domains were predicted
at the N- and C-termini, suggesting that the DzSE protein is
anchored to the membrane. The phylogenetic tree indicated that
SEs were divided into true SEs and SE-like proteins. DzSE was
classified as a true SE and clustered in the same subclade with SE
isolated from Chlorophytum borivilianum (Fig. S3).
The calculated monomeric molecular weight and theoretical pI
of DzCPR were 76.8 kDa and 5.49, respectively. Amino acid sequence
alignment showed that DzCPR had three characteristic domains, the
FAD-, FMN-, and NADPH-binding domains (Fig. S2). The conserved
motif LGDDDQCIEDD in DzCPR was presumed to interact with P450s
and cytochrome c. A linker region between the FAD- and FMN-
binding domains promotes internal electron transfer and assists in
the interaction of CPR with diverse P450s. The phylogenetic tree
revealed that DzCPR belongs to CPR class I and was closed to CPR
isolated from Brachypodium distachyon (Fig. S4).
2.4. Enzyme activity assay
DzCPR enzymatic activity was measured by spectrophotometry
at 550 nm. The reaction contained 1 mg of protein and 50 mM cy-
tochrome c in 1 mL of 0.3 M potassium phosphate buffer (pH 7.6) at
25 ꢀC. The change in the absorbance was recorded after adding
10 mL of 10 mM NADPH solution. Enzyme activity was detected at 1-
min intervals and measured by cytochrome c reduction [19]. To
evaluate the kinetic parameters of cytochrome c, various amounts
of cytochrome c were added to the reactions, while the concen-
tration of NADPH remained fixed at 50 mM. The kinetic parameters
of NADPH were determined using the same principle. The Michaelis
constant Km and Vmax were calculated using nonlinear Michaelis-
Menten plots using GraphPad Prism 7 software.
DzSE enzymatic activity was determined by the synthesis of 2,3-
oxidosqualene. The reaction mixture contained 0.2 mg squalene
(Sigma-Aldrich, USA), 0.5 mg recombinant DzSE protein, 0.5 mg
recombinant DzCPR protein, 1 mM FAD, 1 mM NADPH, Triton X-100
(0.5%), and 50 mM Tris-HCl (pH 7.5) in a total volume of 2 mL. The
mixture was extracted with hexane for three times after incubation
at 25 ꢀC overnight. The extracts were analyzed by gas
chromatography-mass spectrometry (GC-MS).
3.2. Recombinant protein expression in E. coli
The entire DzSE ORF was integrated into the pMal-c2x expres-
sion vector containing the tac-promoter and the maltose-binding
protein (MBP) sequence to generate a fusion protein. The con-
structed pMal-DzSE plasmid was transformed into Transetta (DE3)
chemically competent cells and induced at a low temperature to
reduce the formation of inclusion bodies. After induction, a band at
approximately 98 kDa (55.7-kDa DzSE protein plus the 42.5-kDa
maltose-binding protein) was only partially observed in the su-
pernatant (Fig. S5A). To increase the solubility of the protein, 74
amino acids at the N-terminus of DzCPR were truncated to exclude
a predicted transmembrane region. The remaining sequence was
inserted into the pET-32a vector with expression under the control
of the T7-promoter. SDS-PAGE revealed that the truncated DzCPR
was 86.8 kDa (68.8 kDa DzCPR with an 18-kDa fusion tag) and was
expressed in supernatant (Fig. S5B).
2.5. Subcellular localization
The full-length DzSE and DzCPR open reading frames (ORFs)
were cloned into a modified plant expression vector pCXUN-GFP by
homologous recombination using the ClonExpress II One Step
Cloning kit (Vazyme, China) to allow expression of protein fused
with green fluorescent protein (GFP). The construct vectors DzSE-
GFP and DzCPR-GFP were co-transformed into the rice protoplasts
by PEG-mediated [20]. Transformed protoplasts were visualized
using an Olympus FV1000 confocal microscope.
2.6. Tissue-specific expression and elicitor treatment
To determine tissue expression profiles of DzSE and DzCPR, total
RNA was extracted from young leaves, mature leaves, stems,
flowers, and rhizomes of D. zingiberensis, and reverse transcribed
into cDNA. qRT-PCR primers (Table 1) were designed and the re-
actions were performed using Fast Start Universal SYBR Green
Master (Roche, Germany) with a CFX96 real-time system (Bio-Rad,
USA). The GAPDH housekeeping gene was used as an internal
control. To explore the effects of elicitor treatment, young leaves
3.3. Identification of SE and CPR function
To demonstrate that SE and CPR were catalytically active in 2,3-
oxidosqualene synthesis, we first confirmed CPR activity. DzCPR
enzyme activity and kinetic parameters were measured via
adjusting the concentration of NADPH or cytochrome c in an assay.
In the assay reaction, cytochrome c was reduced after receiving
electrons from NADPH via CPR. The Km and Vmax of DzCPR were
were harvested after treatment with 50 mM methyl jasmonate
(MeJA) at 6, 12, 24, 48, and 72 h for RNA isolation, including a
control. All samples were examined in triplicate and experimental
12.1 0.9
(Fig. 2A), and 15.9 1.3
cytochrome c (Fig. 2B), respectively.
m
M
and 10.7 0.2
m
moL/min/mg protein for NADPH
data were analyzed using the 2DD method.
mM and 10.8 0.3 m
moL/min/mg protein for
Ct
3. Results
DzSE function was determined by GC-MS analysis of the prod-
ucts from the incubation of purified recombinant SE and CPR pro-
teins with squalene and NADPH. The GC results revealed that a new
peak appeared at 11.66 min (Fig. 2C), which was identified as 2,3-
oxidosqualene according to the characteristics of the primary ion
peaks (Fig. 2D). However, 2,3-oxidosqualene was not detected in
the extracts without addition of the CPR protein, even when the
amount of exogenous squalene was increased.
3.1. Cloning and analysis of DzSE and DzCPR gene sequences
A core fragment of each of SE and CPR gene was amplified from
leaf cDNA using degenerate primers. Specific primers were then
designed according to the obtained core sequences to amplify the
50- and 30-terminal sequences. Fragments of SE and CPR genes were
Please cite this article as: W. Song et al., Functional characterization of squalene epoxidase and NADPH-cytochrome P450 reductase in Dioscorea
Song, Wei
