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L.-Y. Song et al. / Biochemical and Biophysical Research Communications 431 (2013) 675–679
from the His boxes of Arabidopsis thaliana FAD2 (
can convert this desaturase into a bifunctional desaturase/hydrox-
ylase [7].
D
12-desaturase)
the Kpn I-Sac I sites of the plasmid pYES2 (Invitrogen, Paisley,
UK). The primers used for cloning the deletion enzyme were CL
and C381R (Table 1). By modifying the sequences of the fusion pro-
tein, we amplified the PCR fragments of the two different desatu-
rases sharing common sequences at their ends using primers CL,
CA381R1 (the PCR fragments was named Fus-CL), CA381L2, and
AR (the PCR fragment was named Fus-AR) (Table 1). In the subse-
quent reaction, Fus-CL and Fus-AR were used as the template, and
CL and AR were used as primers. Fus-CL and Fus-AR formed an
overlap by hybridizing to each other via the common sequences
at their ends. Extension of this overlap by the Pfu DNA polymerase
(Transgen, Beijing, China) yielded a recombinant molecule. The
temperature profile for a typical overlap extension PCR reaction
was 94 °C for 3 min, 94 °C for 30 s, 55 °C for 1 min, 72 °C for
2 min for 35 cycles, and 72 °C for 10 min.
Domain swapping and site-directed mutagenesis were also
widely used in studies of front-end desaturases. Domain deletion
demonstrated that Cytb5 domain was essential for the desaturase
activity, i.e., deleting either the first 112 or 146 residues of the
Cytb5 domain of the borage
of the desaturase activity [8]. Disruption of the his41 of the Cytb5
domain of borage D6D led to the failure to produce
6-fatty acids
D
6-fatty acid desaturase led to the loss
D
[8]. In addition, replacing the first 113 residues of borage D6D with
the residues from Arabidopsis D8D led to the decrease in the cata-
lytic activity, further demonstrating the importance of the Cytb5
domain [9]. Researchers found that the mutation of the Cytb5-like
domain of the rat D6D could not recover its activity in the presence
of other cytochrome b5 proteins, and the free endoplasmic reticu-
lum cytochrome b5 also played a role in the D6D activity [10].
The three conserved His boxes and the residues near them have
been widely investigated. Previous reports indicate that the variant
glutamine in the third histidine box is essential for the D6D activity
and that histidine could not substitute for the glutamine [9,11]. A
mutational study of the conserved amino acid residues of Spirulina
D6D demonstrated that H313 was involved in regioselectivity, i.e.,
2.3. Preparation of site-directed mutations of RnD6C
The template for site-directed mutations was the plasmid pyes-
RnD6C [16]. The preparation of the site-directed mutations of
RnD6C was performed using a QuikChangeÒ Site-Directed Muta-
genesis Kit (Stratagene, La Jolla, Canada). The primers used in the
mutation preparation are listed in Table 2. The PCR reaction tem-
perature profile was 94 °C for 3 min, 94 °C for 30 s, 55 °C for 40 s
and 72 °C for 7 min for 17 cycles. The mutations were amplified
in Eschershia coli, sequenced and then transformed into yeast INV
Sc 1.
The four mutations of C-terminal amino acid residues were pre-
pared using primers listed in Table 3 based on RnD6C sequence.
The temperature profile for the PCR reaction was 94 °C for 3 min,
94 °C for 30 s, 55 °C for 1 min, and 72 °C for 2 min for 35 cycles,
and 72 °C for 10 min.
changing H313 to R313 switched the D6D to
D15 desaturase; that
the three His boxes and H313, H315, D138 and E140 were likely to
be providers of the catalytic Fe center; and that W294 might take
part in forming part of the substrate-binding pocket [12]. The S213
and K218 that are adjacent close to the His box II of Mucor rouxii
D6D were involved in the substrate recognition [13].
The functions of the C-terminal region of the membrane-bound
D
12-fatty acid desaturase and 15-fatty acid desaturase have previ-
D
ously been investigated [14,15]. The results showed that the car-
boxyl terminus of the desaturases had an important function in
determining specific regiochemical and/or substrate characteris-
tics. With regard to the D6D and D8D family, only one report
showed that the C-terminal region might have an important role
in the lipid head group recognition [4,5], but the report lacked de-
2.4. Yeast expression plasmid construction and transformation
2.4.1. Characterization of genes with
Saccharomyces cerevisiae
D6-desaturase function in
tailed information. Blackcurrant
D
6-fatty acid desaturase RnD6C
2.4.1.1. Fatty acid analysis. The methods for these three parts are
previously described elsewhere [16].
and
D
8-sphingolipid desaturase RnD8A have a high sequence sim-
ilarity and a close phylogenetic relationship [16]. Therefore, these
desaturases are suitable materials to study the relationship of
functional region and the regioselectivity of D6D and D8D. In this
study, using domain swapping and site-directed mutagenesis
methods, we found that the region of the C-terminal 67 aa of
D6D was essential and could not be deleted and that several amino
acid residues in that region were involved in the D6D catalytic
activity. The D6D activity of I417T was significantly increased to
132.6 15.6% of RnD6C. However the D6D activity of VK3945AI
was significantly decreased to 36.8 3.01% of RnD6C.
3. Results
3.1. Construction of chimeric genes
Overlap extension PCR method for chimera construction allows
the introduction of junction or mutation sites into the chimeric en-
zymes. There are only 8 different amino acid residue sites in the C-
terminus (after His III) of RnD6C and RnD8A: K389T, VK394, 5AI,
F411L, I417T, IS435, 6VP and Y447H (the letter to the left of the
number represents the amino acid residues in RnD6C, the number
represents the site of the different amino acid residue and the let-
ter on the right of the number represents the amino acid residues
in RnD8A). We deleted the C-terminal 67 aa of RnD6C and named it
RnD6C381 (Fig. 1A); we fused the C-terminal 67 aa of RnD8A into
N-terminal 381 aa of RnD6C and named it RnD6CA381 (Fig. 1A);
we exchanged nine sites of RnD8A into RnD6C and named them
individually as K389T, V394A, K395I, F411L, I417T, S436P,
Y447H, VK3945AI and IS4356VP (Fig. 1B).
2. Materials and methods
2.1. Chemicals and materials
The LA, ALA, GLA and heptadecanoic acid were obtained from
Sigma–Aldrich Trading Co., Ltd. (Shanghai, China). The Pfu DNA
polymerase was obtained from Transgen (Beijing, China). Restric-
tion enzymes were obtained from Takara (Dalian, China). The vec-
tor pYES2 and yeast strain INV Sc 1 were obtained from Invitrogen
(Paisley, UK).
3.2. Functional characterization of the mutations
2.2. Preparation of deletion and fusion mutations of RnD6C
For functional identification, the mutated and the wild-type
desaturases were expressed in S. cerevisiae strain INVScI, and the
recombinant yeast cells were analyzed for fatty acids. Yeast was
also transformed with an empty vector pYES2 as a negative con-
The templates for the deletion and fusion mutation construct
were the plasmid pyes-RnD6C and pyes-RnD8A [16] cloned into
Song, Li-Ying
