J.-U. An et al.
BBA-MolecularandCellBiologyofLipids1863(2018)823–833
horizontal gene transfer because of the symbiotic relationship of these
bacteria with higher organisms [20].
2.3. Cellular fatty acid composition
The N-terminal domain of LOXs is associated with stabilization,
while the C-terminal domain is associated with catalysis, which is
performed by catalytic residues and iron or manganese ion [21–23].
Generally, all LOXs have a common sequence for the catalytic metal-
interacting residues in the C-terminal domain, which are strictly con-
served with HHHNI. The regiospecificity of LOXs is related to the type
of substrate and size of the hydrophobic active-site pocket. A few amino
acids at the bottom of the substrate-binding site are known to be re-
giospecific according to the structures of LOXs. For example, the re-
giospecificity of human ARA 5-LOX and rabbit ARA 15-LOX is asso-
ciated with Phe359, Ala424, Asn425, and Ala603 [24] and Phe353,
Ile418, M419, and Ile593, respectively [25]. The regiospecificity of the
bacterial LOX from P. aeruginosa is thought to involve Glu369, Met434,
Phe435, and Leu612 based on sequence alignment of the regiospecific
residues of rabbit ARA 15-LOX [26]. The regiospecificity of mini-LOX
from Anabaena sp. involves Ala215 [12].
It is important to obtain a LOX with distinct regiospecificity because
the regiospecific-products of LOX are important oxylipins that regulate
physiological activity in vivo in various organisms. Bacterial LOXs are
more active and stable in vitro and expressed more easily and rapidly
than eukaryotic LOXs. Thus, identifying a new regiospecific LOX from
bacterial source is valuable for the biosynthesis of oxylipins. Here, a
LOX with unique regiospecificity was found in the proteobacterium M.
xanthus, which was different with the previously reported LOX from the
same strain [18]. The residues involved in the regiospecificity of M.
xanthus LOX were identified, and a novel variant with altered re-
giospecificity was identified by investigating the regiospecificity of the
variants for these residues based on structural analysis of the homology
model.
Cellular fatty acid composition was measured by the Microbial
Identification Incorporation (MIDI) Sherlock system. The harvested M.
xanthus cells (40 mg) were resuspended in 1 mL of reagent A (50%
methanol containing 150 mg sodium hydroxide), and the mixture was
heated in boiling water on 30 min for saponification. After cooling,
2 mL of reagent B (methanol/hydrochloric acid, 1:1.2 v/v) was added to
the mixture and heated for 10 min at 80 °C for methylation. Fatty acid
methyl esters were extracted with 1.25 mL of reagent C (hexane/methyl
tert-butyl ester, 1:1 v/v) and the aqueous phase was removed. The ex-
tracted fatty acid methyl esters in the solvent phase were analyzed by
GC (Agilent 6890N, Santa Clara, CA, USA) with a flame ionization
detector.
2.4. Culture conditions and enzyme expression
Recombinant E. coli was cultivated at 37 °C in a 2-L flask containing
450 mL of LB medium supplemented with 0.1 mM kanamycin with
shaking at 200 rpm. When the optical density of the bacterial culture at
600 nm reached 0.6, 0.1 mM isopropyl-β-D-thiogalactopyranoside
(IPTG) was added to induce enzyme expression, and the culture was
further incubated at 16 °C with shaking at 150 rpm for 18 h.
Recombinant cells expressing LOX were harvested by centrifugation at
13,000 ×g for 20 min at 4 °C and stored at −80 °C.
2.5. Enzyme purification
The harvested cells were used for enzyme purification as described
previously [17]. The cells were disrupted using a sonicator on ice for
20 min. Unbroken cells and cell debris were removed by centrifugation
at 13,000 ×g for 10 min at 4 °C, and the supernatant was filtered
through a 0.45-μm-pore-size filter. The filtered solution was applied to
an immobilized metal ion affinity chromatography cartridge (Bio-Rad,
Hercules, CA, USA) equilibrated with 50 mM phosphate buffer (pH 8.0)
containing 300 mM NaCl. Bound protein in the cartridge was eluted
with a linear gradient of 10–250 mM imidazole at a flow rate of
1 mL min−1. Active fractions were collected and loaded onto a Bio-Gel
P-6 desalting cartridge (Bio-Rad) equilibrated with 50 mM 3-[4-(2-hy-
2. Materials and methods
2.1. Materials
PUFA standards, including LA, ALA, GLA, ARA, EPA, and DHA, were
purchased from Sigma (St. Louis, MO, USA) and HFA standards, in-
cluding 13-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE), 13-
hydroxy-9,11,15(Z,E,Z)-octadecatrienoic acid (13-HOTrE), 13-hydroxy-
6,9,11(Z,Z,E)-octadecatrienoic acid (13-HOTrEγ), 12-hydroxyei-
cosatetraenoic acid (12-HETE), 15-HETE, 12-hydroxyeicosapentaenoic
acid (12-HEPE), 15-HEPE, 14-hydroxydocosahexaenoic acid (14-
HDOHE), and 17-HDOHE, were purchased from Cayman Chemical
(Ann Arbor, MI, USA).
droxyethyl)-1-piperazinyl]-propanesulfonic
acid
(EPPS)
buffer
(pH 8.5). The loaded protein was eluted using the same buffer at a flow
rate of 1 mL min−1, and the eluted protein was used as the purified
enzyme.
2.6. Determination of molecular mass and metal ion in enzyme
The subunit molecular mass of the putative LOX from M. xanthus
was examined by SDS-PAGE under denaturing conditions using mole-
cular mass marker proteins as reference proteins. The total molecular
mass of the enzyme was determined by gel filtration chromatography
using a Sephacryl S-300 HR 16/60 preparative-grade column (GE
Healthcare, Little Chalfont, UK). The enzyme solution was applied to
the column and eluted with 50 mM Tris-HCl buffer (pH 7.5) containing
150 mM NaCl at a flow rate of 1 mL min−1. The retention time of the
putative LOX from M. xanthus was measured during elution. The
column was calibrated with catalase (206 kDa), aldolase (158 kDa),
conalbumin (75 kDa), ovalbumin (43 kDa), and carbonic anhydrase
(29 kDa) as a gel filtration calibration kit (Amersham Pharmacia
Biotech, now changed to GE healthcare) [27,28]. The total molecular
mass of the enzyme was calculated by comparison with the retention
times of the reference proteins. The contents of metal ions in the pur-
ified putative LOX from M. xanthus were measured by ICP-MS. The
purified LOX with a final concentration of 370 μg mL−1 (4.62 μM) was
prepared. The analysis was performed using NexOn 350D (PerkinElmer
SCIEX, Wellesley, MA, USA) at the NCIRF facility (Seoul National
University, Seoul, Republic of Korea). The instrument was calibrated by
2.2. Bacterial strains, plasmid, gene cloning, and site-directed mutagenesis
M. xanthus DK 1622 (KCCM, Seoul, Republic of Korea), Escherichia
coli ER2566, and pET-28a were used as the source of genomic DNA,
host cells, and expression vector, respectively. Cloning and site-direct
mutagenesis were carried out using primers synthesized by Macrogen
(Seoul, Republic of Korea) (Supplementary Table 1). Primer sequences
with EcoRI and NotI restriction sites designed based on the DNA se-
quence of a putative LOX from M. xanthus (GenBank accession number,
WP_011551854.1) were used for gene cloning. The gene encoding the
putative LOX was amplified by PCR using M. xanthus genomic DNA as
the template and Taq polymerase (Solgent, Daejon, Korea). The DNA
fragment was ligated with the pET-28a vector and transformed into E.
coli ER2566. Recombinant E. coli was plated on Luria-Bertani (LB) agar
containing 0.1 mM kanamycin, an antibiotic resistant colony was se-
lected, and the plasmid DNA sequence was checked by Macrogen. Site-
directed mutagenesis was carried out using the Quick-Change kit
(Stratagene, La Jolla, CA, USA) according to the manufacturer's in-
structions.
824